Mucus clearance is an important airway innate defense mechanism. Airway-targeted overexpression of the epithelial Na(+) channel β-subunit encoded by sodium channel nonvoltage gated 1, beta subunit ...(Scnn1b) in mice Scnn1b-transgenic (Tg) mice increases transepithelial Na(+) absorption and dehydrates the airway surface, which produces key features of human obstructive lung diseases, including mucus obstruction, inflammation, and air-space enlargement. Because the first Scnn1b-Tg mice were generated on a mixed background, the impact of genetic background on disease phenotype in Scnn1b-Tg mice is unknown. To explore this issue, congenic Scnn1b-Tg mice strains were generated on C57BL/6N, C3H/HeN, BALB/cJ, and FVB/NJ backgrounds. All strains exhibited a two- to threefold increase in tracheal epithelial Na(+) absorption, and all developed airway mucus obstruction, inflammation, and air-space enlargement. However, there were striking differences in neonatal survival, ranging from 5 to 80% (FVB/NJ<BALB/cJ<C3H/HeN<C57BL/6N), which correlated with the incidence of upper airway mucus plugging and the levels of Muc5b in bronchoalveolar lavage. The strains also exhibited variable Clara cell necrotic degeneration in neonatal intrapulmonary airways and a variable incidence of pulmonary hemorrhage and lung atelectasis. The spontaneous occurrence of a high surviving BALB/cJ line, which exhibited delayed onset of Na(+) hyperabsorption, provided evidence that: 1) air-space enlargement and postnatal death were only present when Na(+) hyperabsorption occurred early, and 2) inflammation and mucus obstruction developed whenever Na(+) hyperabsorption was expressed. In summary, the genetic context and timing of airway innate immune dysfunction critically determines lung disease phenotype. These mouse strains may be useful to identify key modifier genes and pathways.
The severe diabetic nephropathy that develops in the hypertensive transgenic (mRen-2)27 rat with streptozotocin (STZ) diabetes has previously been considered angiotensin II-dependent. Because ...metabolic pathways are also activated in the diabetic kidney, the present study aimed to determine whether renoprotection could be afforded with inhibitors of advanced glycation end products (AGEs), ALT-946, and aminoguanidine (AG). At 6 weeks of age, nondiabetic control and STZ diabetic Ren-2 rats were randomized to receive vehicle, ALT-946 (1 g/l), or AG (1 g/l) and were studied for 12 weeks. Systolic blood pressure was unchanged with diabetes, ALT-946, or AG. Both kidney weight and glomerular filtration rate were increased with diabetes and unchanged with ALT-946 or AG. ALT-946 and AG equally ameliorated glomerulosclerosis and medullary pathology; however, ALT-946 did reduce cortical tubular degeneration to a greater extent than AG. Albumin excretion rate, which was elevated with diabetes, was reduced with ALT-946 but not AG. AGE immunolabeling was increased in glomeruli and reduced with ALT-946 and AG. These findings indicate that even in the context of renal injury presumed to be primarily blood pressure- and/or angiotensin II-dependent, approaches that interfere with metabolic pathways such as inhibitors of AGE formation can confer renal protection in experimental diabetes.
Changes in podocyte number and morphology have been implicated in the pathogenesis of proteinuria and the progression of human and experimental kidney disease. This study sought to examine podocyte ...foot process and slit pore architecture in experimental diabetic nephropathy and to determine whether such changes were modified with renoprotective intervention by blockade of the renin-angiotensin system.
The number of filtration slits per 100 microm of glomerular basement membrane was assessed by transmission electron microscopy and quantitated histomorphometrically in control animals and in rats with 24 weeks of streptozotocin-induced diabetes. Diabetic rats were either untreated or received the angiotensin converting enzyme inhibitor ramipril, or the angiotensin II type 1 receptor antagonist, valsartan.
When compared with control animals, diabetes was associated with a decrease in the number of slit pores per unit length of glomerular basement membrane, indicative of podocyte foot process broadening. Both ramipril and valsartan attenuated these ultrastructural changes to a similar degree. These differences remained after correcting for glomerular volume as a possible confounding variable.
Preservation of podocyte architecture could contribute to the renoprotective effects of renin-angiotensin system blockade in diabetic nephropathy.
In most aerobic organisms hemoperoxidases play a major role in H2O
2-detoxification, but trypanosomatids have been reported to lack this activity. Here we describe the properties of an ...ascorbate-dependent hemoperoxidase (TcAPX) from the American trypanosome Trypanosoma cruzi. The activity of this plant-like enzyme can be linked to the reduction of the parasite-specific thiol trypanothione by ascorbate in a process that involves nonenzymatic interaction. The role of heme in peroxidase activity was demonstrated by spectral and inhibition studies. Ascorbate could saturate TcAPX activity indicating that the enzyme obeys Michaelis-Menten kinetics. Parasites that overexpressed TcAPX activity were found to have increased resistance to exogenous H2O
2. To determine subcellular location an epitope-tagged form of TcAPX was expressed in T. cruzi, which was observed to colocalize with endoplasmic reticulum resident chaperone protein BiP. These findings identify an arm of the oxidative defense system of this medically important parasite. The absence of this redox pathway in the human host may be therapeutically exploitable.
The finding that the systemic renin-angiotensin system (RAS) is not activated in most types of chronic renal disease has led to the suggestion that a local, intrarenal RAS may be an important ...determinant in the relentless progression of renal disease. Therefore, cell specific changes in various components of the RAS in response to renal mass reduction and angiotensin converting enzyme (ACE) inhibition were examined. Thirty Sprague-Dawley rats were randomly assigned to sham surgery, subtotal nephrectomy (STNx) alone or STNx treated with the ACE inhibitor, perindopril, and sacrificed after 12 weeks. In sham rats, renin mRNA and protein were only present in the juxtaglomerular apparatus. In contrast, in STNx kidneys, renin and angiotensin II expression were noted predominantly in renal tubular epithelial cells in association with overexpression of the prosclerotic cytokine, transforming growth factor-beta1 (TGF-beta1). In perindopril-treated STNx rats expression of renin and TGF-beta1 were similar to control animals. These finding indicate that following renal mass reduction there is pathological tubular expression of various components of the RAS. Furthermore, in contrast to the juxtaglomerular apparatus, tubular renin expression was reduced with ACE inhibition. These changes within the intrarenal RAS may be pathogenetically linked to the development of tubulointerstitial injury.
Detoxification of hydroperoxides in trypanosomes is mediated by a series of linked redox pathways that are dependent on the parasite-specific thiol trypanothione for reducing equivalents. These ...pathways are characterized by differences in subcellular location, electron transport molecules, and substrate specificity. To determine the functional significance of the enzymes involved, we have used a tetracycline-inducible RNA interference system to down-regulate expression of each of the corresponding transcripts in bloodstream form Trypanosoma brucei. We have identified two peroxidases, a cytosolic peroxiredoxin (TbCPX) and a member of the non-selenium glutathione-dependent peroxidase family (TbGPXI), that appear to be essential for the viability of this clinically relevant stage of the parasite life cycle. The addition of tetracycline to the cultures resulted in a major reduction in mRNA levels and enzyme activity, a dramatic fall in growth rate, and significant cell death. Furthermore, within 20 h of adding tetracycline, cells in which the cytosolic peroxiredoxin transcript was targeted were found to be 16-fold more susceptible to killing by exogenous hydrogen peroxide. We also observed that knockdown of the tryparedoxin TbT-PNI, a thioredoxin-like protein that facilitates electron transport to both TbCPX and TbGPXI, resulted in a reduction in growth rate. These experiments therefore identify redox pathways that are essential for oxidative defense in T. brucei and validate the corresponding peroxidases as targets for drug design.
The pathological accumulation of extracellular matrix is a characteristic feature of diabetic cardiomyopathy that is directly related to a loss of function. Tranilast (n-3,4-anthranilic acid), used ...for the treatment of fibrotic skin diseases, has also been shown to inhibit transforming growth factor-beta (TGF-beta)-induced matrix production in kidney epithelial cells.
To investigate the effects of tranilast in the diabetic heart, we examined its effects in cultured cardiac fibroblasts and then assessed its effects in (mRen-2)27 diabetic rats with established disease (8 weeks after streptozotocin).
In vitro studies demonstrated a 58% reduction in TGF-beta1-induced 3H-hydroxyproline incorporation with tranilast 30 microM (p<0.01). At 16 weeks, diabetes in the Ren-2 rat was associated with increased cardiac fibrosis and evidence of TGF-beta1 activation, as measured by the abundance of phosphorylated Smad2. Despite persistent hyperglycaemia and hypertension, tranilast attenuated cardiac fibrosis by 37% (p<0.05) in association with reduction in phospho-Smad2 (p<0.01).
These findings indicate that tranilast has antifibrotic actions in the Ren-2 model of experimental diabetic cardiac disease by mechanisms that might attributable to reduced TGF-beta activity.
A new model of progressive diabetic renal disease in the transgenic (mRen-2)27 rat (TGR).
The tissue renin-angiotensin system (RAS) may modulate the structural and functional changes that occur in ...the diabetic kidney.
Hypertensive transgenic (mREN-2)27 rat (TGR) that exhibit increased tissue renin expression were administered streptozotocin (STZ, diabetic) or citrate buffer (non-diabetic) at six weeks of age, and sacrificed 4 and 12weeks later. Further groups were treated for 12weeks post-STZ or vehicle with the angiotensin converting enzyme inhibitor, perindopril. Comparisons were made with 18-week-old non-diabetic and diabetic spontaneously hypertensive rats (SHR).
In diabetic TGR, the most florid lesion was seen after 12weeks of STZ, with kidneys exhibiting vacuolated tubules, hylanized arterioles, medullary fibrosis and necrosis and severe glomerulosclerosis. In contrast, only mild glomerulosclerosis was seen in non-diabetic TGR and diabetic SHR. Glomerular filtration rate was increased after four weeks of diabetes in TGR and 12weeks of diabetes in SHR, but declined by greater than 50% after 12weeks of diabetes in TGR. In both TGR and SHR, diabetes increased albuminuria but did not modify systolic blood pressure. Renal renin content increased progressively in diabetic TGR, and this was associated with increased renin immunolabeling in the juxtaglomerular apparatus (JGA) and the appearance of renin in proximal convoluted tubules. In contrast, renal renin content and JGA renin immunolabeling were unchanged in diabetic SHR. Perindopril attenuated renal pathology, improved renal function and abolished proximal tubular renin immunolabeling in diabetic TGR.
This is the first report of a diabetic rodent model developing rapid onset renal impairment. Furthermore, this study suggests a role for an activated renal RAS in the acceleration of diabetic renal disease and confirms the benefit of drugs that inhibit this system.
Attenuation of tubular apoptosis by blockade of the renin-angiotensin system in diabetic Ren-2 rats.
Tubular atrophy is a major feature of most renal diseases and is closely associated with loss of ...renal function. The present study sought to investigate tubular epithelial cell apoptosis in experimental diabetic nephropathy and to explore the role of pro-apoptotic transforming growth factor-β (TGF-β) and anti-apoptotic growth factors epidermal growth factor (EGF). The effects of renoprotective therapy with blockade of the renin-angiotensin system (RAS) also were examined.
Six-week-old female Ren-2 rats were injected with streptozotocin (STZ) and maintained diabetic for 12 weeks. Further groups of diabetic rats were treated with the angiotensin-converting enzyme (ACE) inhibitor, perindopril, or the angiotensin II type 1 (AT1) receptor antagonist, valsartan, for 12 weeks.
Widespread apoptosis, identified immunohistochemically by single stranded DNA and TUNEL, was noted in the tubules of diabetic Ren-2 rats. These changes were associated with a 50% decrease in EGF expression and a twofold increase in TGF-β1 mRNA. Treatment of diabetic Ren-2 rats with either valsartan (20 mg/kg/day) or perindopril (6 mg/kg/day) reduced apoptosis to control levels in association with supranormal levels of EGF mRNA (P < 0.01) and a reduction in TGF-β1 gene expression (P < 0.05) to that of control rats.
Tubular apoptosis is a prominent feature of diabetic Ren-2 rats that is attenuated by blockade of the RAS in association with modulation of pro- and anti-apoptotic growth factor expression.
Trypanosoma cruziglutathione-dependent peroxidase I (TcGPXI) can reduce fatty acid, phospholipid, and short chain organic hydroperoxides utilizing a novel redox cycle in which enzyme activity is ...linked to the reduction of trypanothione, a parasite-specific thiol, by glutathione. Here we show that TcGPXI activity can also be linked to trypanothione reduction by an alternative pathway involving the thioredoxin-like protein tryparedoxin. The presence of this new pathway was first detected using dialyzed soluble fractions of parasite extract. Tryparedoxin was identified as the intermediate molecule following purification, sequence analysis, antibody studies, and reconstitution of the redox cycle in vitro. The system can be readily saturated by trypanothione, the rate-limiting step being the interaction of trypanothione with the tryparedoxin. Both tryparedoxin and TcGPXI operate by a ping-pong mechanism. Overexpression of TcGPXI in transfected parasites confers increased resistance to exogenous hydroperoxides. TcGPXI contains a carboxyl-terminal tripeptide (ARI) that could act as a targeting signal for the glycosome, a kinetoplastid-specific organelle. Using immunofluorescence, tagged fluorescent proteins, and biochemical fractionation, we have demonstrated that TcGPXI is localized to both the glycosome and the cytosol. The ability of TcGPXI to use alternative electron donors may reflect their availability at the corresponding subcellular sites.
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