Progressive diabetic nephropathy in the Ren-2 rat Kelly, Darren J; Wilkinson-Berka, Jennifer L; Gilbert, Richard E
American journal of physiology. Renal physiology,
05/2007, Letnik:
292, Številka:
5
Journal Article
We previously showed the rat thymus contains and secretes active renin. However, the cellular location of the thymic renin–angiotensin system (RAS) is unknown. To more easily study the thymic RAS we ...used the hypertensive transgenic (mRen-2)27 rat which overexpresses renin and angiotensin in extra-renal tissues. Comparisons were made with normotensive Sprague Dawley (SD) rats. All rats exhibited intense immunolabeling for renin protein and angiotensin in macrophages and thymic epithelial cells, however renin prosequence was not detected. In each rat strain, thymic renin was predominately active and highest in Ren-2 rats (Ren-2, 22.6±4.2, SD 0.8±0.1 mGoldblatt Units/g, mean±SEM). Renin mRNA was identified in Ren-2 and SD rat thymus by RT-PCR. Thymic angiotensin II concentrations/wet weight in Ren-2 (20.1±1.1 fmol/g) and SD (15.8±2.3 fmol/g) rats were similar to plasma. In conclusion, macrophages and epithelial cells are the source of active renin in the rat thymus. The thymic RAS may have actions systemically and may also influence local processes such as blood flow and cell growth.
Background and Aim: Tubular atrophy is a major feature of most renal diseases and is closely associated with the loss of renal function. The present study sought to investigate whether ...Fas/FasL‐induced tubular epithelial cell apoptosis was a feature of experimental diabetic nephropathy. The effects of renoprotective therapy with blockade of the renin‐angiotensin (RAS) system were also examined.
Method: Six‐week‐old female Ren‐2 rats were injected with streptozotocin and maintained diabetic for 12 weeks. Further groups of diabetic rats were treated with the angiotensin‐converting enzyme inhibitor, perindopril, for 12 weeks.
Results: Widespread apoptosis, identified by using mediated Terminal dUTP nick‐end labelling (TUNEL) staining was noted in the tubules of diabetic Ren‐2 rats. These changes were associated with an increase in both Fas mRNA and Fas L (ligand) within the tubules (P < 0.01). Treatment of diabetic Ren‐2 rats with perindopril (6 mg/kg per day) reduced the apoptosis to control levels and was associated with a reduction in Fas mRNA and Fas L protein (P < 0.05).
Conclusion: In conclusion, Fas/Fas L‐induced tubular apoptosis is a feature of diabetic Ren‐2 rats and is attenuated by the blockade of the RAS.
The parasitic protozoan
Trypanosoma cruzi is exposed to toxic oxygen metabolites which arise from drug metabolism or immune mechanisms, in addition to those produced by endogenous processes. ...Identification and functional analysis of parasite enzymes which confer protection against oxidative stress is therefore of importance. To investigate the role of
T. cruzi superoxide dismutase (SOD) we transfected epimastigotes with an expression vector containing a putative Fe-SOD gene homologue and achieved overexpression of enzyme activity (5–8 fold). Inhibition studies carried out on the partially purified enzyme revealed azide and H
2O
2 sensitivity and cyanide insensitivity, the profile expected of an Fe-isoform. Phenotypic analysis of transformed parasites showed that they were more susceptible than control cells to growth inhibition by the trypanocidal drug benznidazole and by gentian violet, an agent which can be used to decontaminate blood supplies in endemic areas. These results may reflect an imbalance in the antioxidant defences of the parasite produced as a result of overexpression of Fe-SOD.
Abstract Preterm infants have a reduced pulmonary diffusing capacity that has been invoked to explain rapid arterial O2 -desaturation during apnea, despite little evidence to support this view. We ...explored the role of diffusion limitation on O2 -desaturation during apnea by developing a mathematical model of gas exchange in which O2 dynamically loads the blood traversing the pulmonary capillary. While normal diffusing capacity ( D L O 2 ) had negligible impact on apneic desaturation, reduced D L O 2 advanced the onset of desaturation during apnea. Unexpectedly, despite considerable diffusion limitation, its influence on O2 -desaturation disappeared within 15 s, because its impact in slowing alveolar O2 depletion maintained a higher driving pressure for diffusion. In contrast, reduced D L O 2 substantially slowed reoxygenation following apnea. Our findings do not support the hypothesis that reduced D L O 2 explains the rapid apneic desaturation observed in preterm infants. Instead, the signature of reduced D L O 2 is a prolonged hypoxemia following apnea, potentially causing a persistence of hypoxic conditions when heart rate and cardiac workload reach a peak.
The role of HLA phenotype as a risk factor for drug-induced gingival overgrowth was investigated in a cohort of 172 transplant recipients. Clinically significant overgrowth warranting surgical ...correction was observed in 72 patients (42%). Using stepwise regression modelling, 6 clinical parameters were identified as significant risk factors for the severity of gingival overgrowth. These were; age, sex, creatinine plasma level, duration of therapy, papilla bleeding index and concomitant medication with a calcium channel blocking drug. 3 HLA alleles were also identified as risk factors when adjusted for other clinically significant risk factors (HLA -DR2, A24, B37). However, when the p-values for the HLA variables were corrected to compensate for the use of multiple significance testing, only HLA-B37 remained statistically significant at the 5% level. Organ transplant patients are at risk of developing gingival overgrowth, with approximately 25% medicated with cyclosporin alone requiring corrective gingival surgery. This figure more than doubles in patients concomitantly medicated with a calcium blocking drug. The data at present available would suggest that the severity of gingival overgrowth is also significantly associated with the HLA-B37 phenotype.
Growth of Trypanosoma cruzi as colonies on solid medium has not been widely used as an experimental procedure. We therefore sought to establish a reliable and routine plating method. The optimal ...results were achieved with a matrix of 0.65% low melting point agarose onto which epimasigotes from the mid-to-late logarithmic phase of growth were spread. Colonies could be isolated after incubation for 21 days in a humidified 5% CO2 environment at 28 degrees C. Plating efficiencies in the range of 40% were obtained by this method and clones could be recovered into liquid medium or onto blood-agar slopes with a high success rate. The procedure has also been adapted for the isolation of genetically transformed clones after electroporation of epimastigotes with either plasmid or cosmid vectors. This was best achieved by inclusion of the electroporated cell inoculum in a 0.6% agarose overlay containing G418 as the selective drug, on top of a 0.8% agar base. Transformation efficiencies were as high as 10(-5) cells per microgram of DNA. A reliable plating method for T. cruzi will have many applications and is a significant step towards the use of 'shotgun transformation' to generate libraries of T. cruzi recombinants.
Superoxide dismutases (SOD) are a family of antioxidant enzymes that function by removing superoxide anions from the cellular environment. Here, we show that the African trypanosome,
Trypanosoma ...brucei, expresses four SOD isoforms, three of which we have validated biochemically as iron dependent, a feature normally associated with prokaryotic SODs. Localisation studies reveal that two of the enzymes are found predominantly in a parasite-specific organelle, the glycosome (TbSODB1 and TbSODB2), while the other two are targeted to the mitochondrion (TbSODA and TbSODC). Functional analysis of the SOD repertoire in bloodstream form parasites was performed using an inducible RNA interference (RNAi) approach. Down-regulation of the glycosomal SOD transcripts corresponded with a significant reduction in the corresponding proteins and a dramatic level of cell death within the population. The importance of one of the mitochondrial enzymes (TbSODA) only became apparent when parasites were exposed to the superoxide-generating agent paraquat following induction of RNAi. These experiments therefore identify essential components of the superoxide metabolising arm of the
T. brucei oxidative defence system and validate these enzymes as parasite-specific targets for drug design.
WD (tryptophan/aspartic acid) repeat proteins perform a wide variety of functions in eukaryotic cells. They are characterised by the presence of a number of conserved repeat motifs that contribute to ...the β-propeller structures which are the common feature of this large group of proteins. We report here the properties of the first characterised member of this family in the American trypanosome,
Trypanosoma cruzi (TcBPP1). In the CL Brener clone the protein is 482 amino acids long and is predicted to contain four WD repeat motifs, flanked by amino and carboxyl terminal extensions.
TcBPP1 is a single copy gene present on a 1.0/1.6 Mb pair of homologous chromosomes in a locus that is syntenic with the corresponding regions of
Trypanosoma brucei and
Leishmania major chromosomes. Consistent with the proposed hybrid nature of the CL Brener clone, the proteins encoded by the two different alleles share only 97% identity at the amino acid level. To determine subcellular location, we examined transfected parasites for the distribution of green fluorescent protein (GFP) fused with different regions of TcBPP1. These studies demonstrated that a 115 amino acid peptide derived from the amino terminal domain of TcBPP1 is able to target GFP to the mitochondrion. Interestingly this region lacks a typical amino terminal presequence suggesting that mitochondrial import is mediated by an alternative targeting signal.
The capacity to synthesize vitamin C (ascorbate) is widespread in eukaryotes but is absent from humans. The last step in the biosynthetic pathway involves the conversion of an aldonolactone substrate ...to ascorbate, a reaction catalyzed by members of an FAD-dependent family of oxidoreductases. Here we demonstrate that both the African trypanosome, Trypanosoma brucei, and the American trypanosome, Trypanosoma cruzi, have the capacity to synthesize vitamin C and show that this reaction occurs in a unique single-membrane organelle, the glycosome. The corresponding T. brucei flavoprotein (TbALO) obeys Michaelis-Menten kinetics and can utilize both L-galactono-γ-lactone and D-arabinono-γ-lactone as substrate, properties characteristic of plant and fungal enzymes. We could detect no activity toward the mammalian enzyme substrate L-gulono-γ-lactone. TbALO null mutants (bloodstream form) were found to display a transient growth defect, a trait that was enhanced when they were cultured in medium in which the essential serum component had been pretreated with ascorbate oxidase to deplete vitamin C. It is implicit, therefore, that bloodstream-form trypanosomes also possess a capacity for ascorbate transport.
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