Quantitative imaging of selenoprotein with multi-isotope imaging mass spectrometry (MIMS).

Multi-isotope imaging mass spectrometry (MIMS) allows high resolution quantitative imaging of protein and nucleic acid synthesis at the level of a single cell using stable isotope labels. We employed MIMS to determine the compartmental localization of selenoproteins tagged with stable isotope selenium compounds in human aortic endothelial cells (HAEC), and to compare the efficiency of labeling (to determine the ideal selenium source) from these compounds: [82Se]-selenite, [77Se]-seleno-methionine, and [76Se]-methyl-selenocysteine. We found that all three selenium sources appear to be localized in the nucleus as well as in the cytoplasm in HAEC. Seleno-methionine appears to be a better source for (seleno)protein synthesis. For MIMS detection, we compared freeze-drying to thin layer vs. thin sectioning for sample preparation. MIMS provides a unique and novel way to dissect selenoprotein synthesis in cells.

S-nitrosothiols and the S-nitrosoproteome of the cardiovascular system.

SIGNIFICANCE: Since their discovery in the early 1990's, S-nitrosylated proteins have been increasingly recognized as important determinants of many biochemical processes. Specifically, S-nitrosothiols in the cardiovascular system exert many actions, including promoting vasodilation, inhibiting platelet aggregation, and regulating Ca(2+) channel function that influences myocyte contractility and electrophysiologic stability. RECENT ADVANCES: Contemporary developments in liquid chromatography-mass spectrometry methods, the development of biotin- and His-tag switch assays, and the availability of cyanide dye-labeling for S-nitrosothiol detection in vitro have increased significantly the identification of a number of cardiovascular protein targets of S-nitrosylation in vivo. CRITICAL ISSUES: Recent analyses using modern S-nitrosothiol detection techniques have revealed the mechanistic significance of S-nitrosylation to the pathophysiology of numerous cardiovascular diseases, including essential hypertension, pulmonary hypertension, ischemic heart disease, stroke, and congestive heart failure, among others. FUTURE DIRECTIONS: Despite enhanced insight into S-nitrosothiol biochemistry, translating these advances into beneficial pharmacotherapies for patients with cardiovascular diseases remains a primary as-yet unmet goal for investigators within the field.

Both selenium deficiency and modest selenium supplementation lead to myocardial fibrosis in mice via effects on redox-methylation balance.

SCOPE: Selenium has complex effects in vivo on multiple homeostatic mechanisms such as redox balance, methylation balance, and epigenesis, via its interaction with the methionine-homocysteine cycle. In this study, we examined the hypothesis that selenium status would modulate both redox and methylation balance and thereby modulate myocardial structure and function. METHODS AND RESULTS: We examined the effects of selenium-deficient (<0.025 mg/kg), control (0.15 mg/kg), and selenium-supplemented (0.5 mg/kg) diets on myocardial histology, biochemistry and function in adult C57/BL6 mice. Selenium deficiency led to reactive myocardial fibrosis and systolic dysfunction accompanied by increased myocardial oxidant stress. Selenium supplementation significantly reduced methylation potential, DNA methyltransferase activity and DNA methylation. In mice fed the supplemented diet, inspite of lower oxidant stress, myocardial matrix gene expression was significantly altered resulting in reactive myocardial fibrosis and diastolic dysfunction in the absence of myocardial hypertrophy. CONCLUSION: Our results indicate that both selenium deficiency and modest selenium supplementation leads to a similar phenotype of abnormal myocardial matrix remodeling and dysfunction in the normal heart. The crucial role selenium plays in maintaining the balance between redox and methylation pathways needs to be taken into account while optimizing selenium status for prevention and treatment of heart failure.

Glutathione peroxidase-3 deficiency promotes platelet-dependent thrombosis in vivo.

BACKGROUND: Glutathione peroxidase-3 (GPx-3) is a selenocysteine-containing plasma protein that scavenges reactive oxygen species in the extracellular compartment. A deficiency of this enzyme has been associated with platelet-dependent thrombosis, and a promoter haplotype with reduced function has been associated with stroke risk. METHODS AND RESULTS: We recently developed a genetic mouse model to assess platelet function and thrombosis in the setting of GPx-3 deficiency. The GPx-3((-/-)) mice showed an attenuated bleeding time and an enhanced aggregation response to the agonist ADP compared with wild-type mice. GPx-3((-/-)) mice displayed increased plasma levels of soluble P-selectin and decreased plasma cyclic cGMP compared with wild-type mice. ADP infusion-induced platelet aggregation in the pulmonary vasculature produced a more robust platelet activation response in the GPx-3((-/-)) than wild-type mice; histological sections from the pulmonary vasculature of GPx-3((-/-)) compared with wild-type mice showed increased platelet-rich thrombi and a higher percentage of occluded vessels. Cremaster muscle preparations revealed endothelial dysfunction in the GPx-3((-/-)) compared with wild-type mice. With a no-flow ischemia-reperfusion stroke model, GPx-3((-/-)) mice had significantly larger cerebral infarctions compared with wild-type mice and platelet-dependent strokes. To assess the neuroprotective role of antioxidants in this model, we found that manganese(III) meso-tetrakis(4-benzoic acid)porphyrin treatment reduced stroke size in GPx-3((-/-)) mice compared with vehicle-treated controls. CONCLUSIONS: These findings demonstrate that GPx-3 deficiency results in a prothrombotic state and vascular dysfunction that promotes platelet-dependent arterial thrombosis. These data illustrate the importance of this plasma antioxidant enzyme in regulating platelet activity, endothelial function, platelet-dependent thrombosis, and vascular thrombotic propensity.

Angiotensin II and hypertonicity modulate proximal tubular aquaporin 1 expression.

Aquaporin 1 (AQP1) is the major water channel in the renal proximal tubule (PT) and thin descending limb of Henle, but its regulation remains elusive. Here, we investigated the effect of ANG II, a key mediator of body water homeostasis, on AQP1 expression in immortalized rat proximal tubule cells (IRPTC) and rat kidney. Real-time PCR on IRPTC exposed to ANG II for 12 h revealed a biphasic effect AQP1 mRNA increased dose dependently in response to 10(-12) to 10(-8) M ANG II but decreased by 50% with 10(-7) M ANG II. The twofold increase of AQP1 mRNA in the presence of 10(-8) M ANG II was abolished by the AT(1) receptor blocker losartan. Hypertonicity due to either NaCl or mannitol also upregulated AQP1 mRNA by three- and twofold, respectively. Immunocytochemistry and Western blotting revealed a two- to threefold increase in AQP1 protein expression in IRPTC exposed concomitantly to ANG II (10(-8)M) and hypertonic medium (either NaCl or mannitol), indicating that these stimuli were not additive. Three-dimensional reconstruction of confocal images suggested that AQP1 expression was increased by ANG II in both the apical and basolateral poles of IRPTC. In vivo studies showed that short-term ANG II infusion had a diuretic effect, while this effect was attenuated after several days of ANG II infusion. After 10 days, we observed a twofold increase in AQP1 expression in the PT and thin descending limb of Henle of ANG II-infused rats that was abolished when rats were treated with the selective AT(1)-receptor antagonist olmesartan. Thus ANG II increases AQP1 expression in vitro and in vivo via direct interaction with the AT(1) receptor, providing an important regulatory mechanism to link PT water reabsorption to body fluid homeostasis via the renin-angiotensin system.

Regulation of the extracellular antioxidant selenoprotein plasma glutathione peroxidase (GPx-3) in mammalian cells.

Plasma glutathione peroxidase (GPx-3) is a selenocysteine-containing extracellular antioxidant protein that catalyzes the reduction of hydrogen peroxide and lipid hydroperoxides. Selenoprotein expression involves the alternate recognition of a UGA codon as a selenocysteine codon and requires signals in the 3'-untranslated region (UTR), including a selenocysteine insertion sequence (SECIS), as well as specific translational cofactors. To ascertain regulatory determinants of GPx-3 expression and function, we generated recombinant GPx-3 (rGPX-3) constructs with various 3'-UTR, as well as a Sec73Cys mutant. In transfected Cos7 cells, the Sec73Cys mutant was expressed at higher levels than the wild type rGPx-3, although the wild type rGPx-3 had higher specific activity, similar to plasma purified GPx-3. A 3'-UTR with only the SECIS was insufficient for wild type rGPx-3 protein expression. Selenocompound supplementation and co-transfection with SECIS binding protein 2 increased wild type rGPx-3 expression. These results demonstrate the importance of translational mechanisms in GPx-3 expression.

Aldosterone increases oxidant stress to impair guanylyl cyclase activity by cysteinyl thiol oxidation in vascular smooth muscle cells.

Hyperaldosteronism is associated with impaired endothelium-dependent vascular reactivity owing to increased reactive oxygen species and decreased bioavailable nitric oxide (NO(.)); however, the effects of aldosterone on vasodilatory signaling pathways in vascular smooth muscle cells (VSMC) remain unknown. Soluble guanylyl cyclase (GC) is a heterodimer that is activated by NO(.) to convert cytosolic GTP to cGMP, a second messenger required for normal VSMC relaxation. Here, we show that aldosterone (10(-9)-10(-7) mol/liter) diminishes GC activity by activating NADPH oxidase in bovine aortic VSMC to increase reactive oxygen species levels and induce oxidative posttranslational modification(s) of Cys-122, a beta(1)-subunit cysteinyl residue demonstrated previously to modulate NO(.) sensing by GC. In VSMC treated with aldosterone, Western immunoblotting detected evidence of GC beta(1)-subunit disulfide bonding, whereas mass spectrometry analysis of a homologous peptide containing the Cys-122-bearing sequence exposed to conditions of increased oxidant stress confirmed cysteinyl sulfinic acid (m/z 435), sulfonic acid (m/z 443), and disulfide (m/z 836) bond formation. The functional effect of these modifications was examined by transfecting COS-7 cells with wild-type GC or mutant GC containing an alanine substitution at Cys-122 (C122A). Exposure to aldosterone or hydrogen peroxide (H(2)O(2)) significantly decreased cGMP levels in cells expressing wild-type GC. In contrast, aldosterone or H(2)O(2) did not influence cGMP levels in cells expressing the mutant C122A GC, confirming that oxidative modification of Cys-122 specifically impairs GC activity. These findings demonstrate that pathophysiologically relevant concentrations of aldosterone increase oxidant stress to convert GC to an NO(.)-insensitive state, resulting in disruption of normal vasodilatory signaling pathways in VSMC.

High glucose stimulates angiotensinogen gene expression and cell hypertrophy via activation of the hexosamine biosynthesis pathway in rat kidney proximal tubular cells.

The present study investigated whether activation of the hexosamine biosynthesis pathway might mediate at least in part the high glucose effect on angiotensinogen (ANG) gene expression and immortalized renal proximal tubular cell (IRPTC) hypertrophy. IRPTC were cultured in monolayer. ANG, renin, and beta-actin mRNA expression were determined by specific RT-PCR assays. Phosphorylation of p38 MAPK, activating transcription factor-2 (ATF-2), and cAMP-responsive element-binding protein (CREB) was determined by Western blot analysis. Cell hypertrophy was assessed by flow cytometry, intracellular p27kip1 protein levels, and [3H]leucine incorporation into proteins. Glucosamine stimulated ANG and renin mRNA expression and enhanced p38 MAPK, ATF-2, and CREB phosphorylation in normal glucose (5 mm) medium. Azaserine and 6-diazo-5-oxo-l-norleucine (inhibitors of glutamine: fructose-6-phosphate amino transferase enzyme) blocked the stimulatory effect of high glucose, but not that of glucosamine, on ANG gene expression in IRPTCs. SB 203580 (a specific p38 MAPK inhibitor) attenuated glucosamine action on ANG gene expression as well as p38 MAPK and ATF-2 phosphorylation, but not that of CREB. GF 109203X and calphostin C (inhibitors of protein kinase C) blocked the effect of glucosamine on ANG gene expression and CREB phosphorylation, but had no impact on p38 MAPK and ATF-2 phosphorylation. Finally, both glucosamine and high glucose induced IRPTC hypertrophy. The hypertrophic effect of glucosamine was blocked in the presence of GF 109203X, but not azaserine and SB 203580. In contrast, the hypertrophic effect of high glucose was blocked in the presence of azaserine and GF 109203X, but not SB203580. Our studies demonstrate that the stimulatory effect of high glucose on ANG gene expression and IRPTC hypertrophy may be mediated at least in part via activation of hexosamine biosynthesis pathway signaling.

Heme: a novel inducer of MCP-1 through HO-dependent and HO-independent mechanisms.

This study examined the effect of hemin on the expression of heme oxygenase-1 (HO-1) and monocyte chemoattractant protein-1 (MCP-1) in immortalized rat proximal tubular epithelial cells (IRPTCs). Hemin elicited a dose- and time-dependent induction of HO-1 and MCP-1 mRNA. HO activity contributed to MCP-1 mRNA expression at early time points (4-6 h) because inhibition of HO activity by zinc protoporphyrin (ZnPP) prevented hemin-induced expression of MCP-1 mRNA. Catalytically active intracellular iron was markedly increased in hemin-treated IRPTCs and contributed to the induction of HO-1 and MCP-1 mRNA because an iron chelator blocked hemin-induced upregulation of both genes, whereas a cell-permeant form of iron directly induced these genes. N-acetylcysteine completely blocked hemin-induced expression of HO-1 and MCP-1 mRNA, thereby providing added evidence for redox regulation of expression of these genes. The redox-sensitive transcription factor NF-kappaB was recruited in hemin-induced upregulation of MCP-1 because two different compounds that abrogate the activation of NF-kappaB (TPCK and BAY 11-7082) completely blocked hemin-induced upregulation of MCP-1 mRNA. In contrast to this HO-mediated induction of MCP-1 through redox-sensitive, iron-dependent, and NF-kappaB-involved pathways observed after 4-6 h, hemin also elicited a delayed induction of MCP-1 at 18 h through HO-independent pathways. We conclude that hemin is a potent inducer of MCP-1 in IRPTCs: HO-dependent, heme-degrading pathways lead to an early, robust, and self-remitting induction of MCP-1, whereas HO-independent mechanisms lead to a delayed expression of MCP-1.

Growth factor-mediated phosphorylation of proapoptotic BAD reduces tubule cell death in vitro and in vivo.

BACKGROUND: Exogenous growth factors administered during unilateral ureteral obstruction (UUO) in neonatal rats significantly reduce apoptosis and tubular atrophy. Because the mechanism underlying these salutary effects is largely unknown, we investigated signaling pathways potentially activated by growth factors to determine their roles in therapeutic action. METHODS: Mechanical strain was applied to confluent cultures of immortalized rat proximal tubule cells to simulate obstruction-induced stretch injury in vivo. Growth factors, inhibitory antibodies or pharmacological inhibitors were added to cultures that were subsequently processed for TUNEL analysis or immunoblots to identify signaling pathways that could be modulating cell survival. For in vivo studies, kidneys harvested from rats +/- UUO +/- epidermal growth factor (EGF) were fixed or frozen for immunohistochemistry or immunoblot analysis. RESULTS: Treatment with EGF or insulin-like growth factor-1 (IGF-1) during stretch decreased apoptosis by 50% (P < 0.001). Neutralizing antibodies (Abs) directed against either growth factor or its receptor blocked the reduction in apoptosis. Stretch decreased BAD phosphorylation by approximately 50% (P < 0.001) relative to unstretched cells and each growth factor restored phosphorylation to basal levels. Kinase-specific inhibitors that blocked growth factor-mediated BAD phosphorylation promoted apoptosis in vitro. BAD phosphorylation decreased by approximately 50% (P < 0.001) in the tubules of obstructed hydronephrotic rat kidneys and administration of EGF restored BAD phosphorylation to basal levels. CONCLUSIONS: Signaling pathways converging at BAD phosphorylation are key to growth factor-mediated attenuation of stretch-induced apoptosis in vitro and in vivo.

Insulin inhibits dexamethasone effect on angiotensinogen gene expression and induction of hypertrophy in rat kidney proximal tubular cells in high glucose.

The present studies investigated whether insulin inhibits the stimulatory effect of dexamethasone (DEX) on angiotensinogen (ANG) gene expression and induction of hypertrophy in rat immortalized renal proximal tubular cells (IRPTCs) in a high-glucose milieu. Rat IRPTCs were cultured in monolayer. ANG and ANG mRNA expression in IRPTCs were quantified by a specific RIA for rat ANG and by RT-PCR assay, respectively. A fusion gene containing the full length of the 5'-flanking region of the rat ANG gene linked to a chloramphenicol acetyl transferase reporter gene was introduced into IRPTCs. The level of fusion gene expression was determined by cellular chloramphenicol acetyl transferase enzymatic activity. Cellular hypertrophy was assessed by flow cytometry, cellular p27(Kip1) protein expression, and protein assay. Our results showed that high glucose (i.e. 25 mM) and DEX (10(-7) M) additively stimulated ANG gene expression and induced IRPTC hypertrophy. Insulin inhibited the effect of high glucose and DEX on these parameters. The inhibitory effect of insulin was reversed by PD 98059 (a MAPK inhibitor) but not by wortmannin (a phosphatidylinositol-3-kinase inhibitor). These results demonstrate that insulin is effective in blocking the stimulatory action of high glucose and DEX on ANG gene expression and induction of IRPTC hypertrophy, suggesting its important role in preventing local intrarenal renin-angiotensin system activation and renal proximal tubular cell hypertrophy induced by hyperglycemia and glucocorticoids in vivo.

High glucose stimulates angiotensinogen gene expression via reactive oxygen species generation in rat kidney proximal tubular cells.

The present studies investigated whether the effect of high glucose levels on angiotensinogen (ANG) gene expression in kidney proximal tubular cells is mediated via reactive oxygen species (ROS) generation and p38 MAPK activation. Rat immortalized renal proximal tubular cells (IRPTCs) were cultured in monolayer. Cellular ROS generation and p38 MAPK phosphorylation were assessed by lucigenin assay and Western blot analysis, respectively. The levels of immunoreactive rat ANG secreted into the media and cellular ANG mRNA were determined by a specific RIA and RT-PCR, respectively. High glucose (25 mM) evoked ROS generation and p38 MAPK phosphorylation as well as stimulated immunoreactive rat ANG secretion and ANG mRNA expression in IRPTCs. These effects of high glucose were blocked by antioxidants (taurine and tiron), inhibitors of mitochondrial electron transport chain complex I (rotenone) and II (thenoyltrifluoroacetone), an inhibitor of glycolysis-derived pyruvate transport into mitochondria (alpha-cyano-4-hydroxycinnamic acid), an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenylhydrazone), a manganese superoxide dismutase mimetic, catalase, and a specific inhibitor of p38 MAPK (SB 203580), but were not affected by an inhibitor of the malate-aspartate shuttle (aminooxyacetate acid). Hydrogen peroxide (>/=10(-5) M) also stimulated p38 MAPK phosphorylation, ANG secretion, and ANG mRNA gene expression, but its stimulatory effect was blocked by catalase and SB 203580. These studies demonstrate that the stimulatory action of high glucose on ANG gene expression in IRPTCs is mediated at least in part via ROS generation and subsequent p38 MAPK activation.

Monitoring changes in gene expression in renal ischemia-reperfusion in the rat.

BACKGROUND: Although acute renal failure (ARF) is a relatively common disorder with major morbidity and mortality, its molecular basis remains incompletely defined. The present study examined global gene expression in the well-characterized ischemia-reperfusion model of ARF using DNA microarray technology. METHODS: Male Wistar rats underwent bilateral renal ischemia (30 min) or sham operation, followed by reperfusion for 1, 2, 3 or 4 days. Plasma creatinine increased approximately fivefold over baseline, peaking on day 1. Renal total RNA was used to probe cDNA microarrays. RESULTS: Alterations in expression of 18 genes were identified by microarray analysis. Nine genes were up-regulated (ADAM2, HO-1, UCP-2, and thymosin beta4 in the early phase and clusterin, vanin1, fibronectin, heat-responsive protein 12 and FK506 binding protein in the established phase), whereas another nine were down-regulated (glutamine synthetase, cytochrome p450 IId6, and cyp 2d9 in the early phase and cyp 4a14, Xist gene, PPARgamma, alpha-albumin, uromodulin, and ADH B2 in the established phase). The identities of these 18 genes were sequence-verified. Changes in gene expression of ADAM2, cyp2d6, fibronectin, HO-1 and PPARgamma were confirmed by quantitative real-time polymerase chain reaction (PCR). ADAM2, cyp2d6, and PPARgamma have not previously been known to be involved in ARF. CONCLUSION: Using DNA microarray technology, we identified changes in expression of 18 genes during renal ischemia-reperfusion injury in the rat. We confirmed changes in five genes (fibronectin, ADAM2, cyp 2d6, HO-1 and PPARgamma) by quantitative real-time PCR. Several genes, not previously been identified as playing a role in ischemic ARF, may have importance in this disease.

Global analysis of gene expression in renal ischemia-reperfusion in the mouse.

Ischemia-induced acute renal failure (ARF) is a relatively common disorder with major morbidity and mortality. To study global gene expression during ARF, 6-week-old C57BL/6 male mice underwent 30 min of bilateral renal ischemia followed by reperfusion [I/R] or sham operation. Oligonucleotide microarrays [Affymetrix] with approximately 10,000 genes, 6,643 of which were present in mouse kidney, were used to analyze mRNA expression for up to 4 days following I/R. Fifty-two genes at day 1 and 40 at day 4 were up-regulated more than 4-fold [400%]. Seventy genes at day 1 and 30 genes at day 4 were down-regulated to under 0.25-fold from baseline [25%]. Real-time quantitative RT-PCR confirmed changes in expression for 8 genes of interest. Most of the induced transcripts are involved in cell structure, extracellular matrix, intracellular calcium binding, and cell division/differentiation. Our data identified several novel genes that may be important in renal repair after ischemia.

Regulation and localization of HSP70 and HSP25 in the kidney of rats undergoing long-term administration of angiotensin II.

Various renal insults result in induction of heat shock protein (HSP) expression within the kidney. Some of the HSPs induced in that manner are postulated to have renoprotective effects via either chaperoning actions or antioxidative properties. We have previously reported that long-term angiotensin (Ang) II administration induces the expression of renal HSP32, also known as heme oxygenase-1 (HO-1). Here, we investigated the regulation of expression and localization of other HSPs, including HSP70, HSP25, and alphaB-crystallin, in the kidney of rats undergoing long-term administration of Ang II (0.7 mg. kg(-1). d(-1)). Immunoblot analysis demonstrated that Ang II increased renal expression of HSP70 and HSP25, as well as HO-1, but that expression of alphaB-crystallin was unaffected by this treatment. The Ang II-induced increase in renal HSP70 and HSP25 was dependent on the angiotensin type 1 receptor activation but not on hypertension per se. Immunohistochemistry revealed that HSP70 and HSP25 were expressed in the medullar regions and in the renal arterial wall in the kidney of control rats. After Ang II infusion, signals for HSP70, HSP25, and HO-1 proteins increased in intensity in the endothelium and medial smooth muscle of the renal artery. In addition, all of these HSPs were induced in proximal renal tubular epithelial cells from the same segments, suggesting that similar mechanisms are responsible for upregulating these HSPs. Our data show that Ang II infusion induces renal HSP70 and HSP25, as well as HO-1, and that Ang II can induce expression of these HSPs in renal cells in a pressor-independent manner.

Abnormal iron deposition in renal cells in the rat with chronic angiotensin II administration.

Acute experimental iron loading causes iron to accumulate in the renal tissue. The accumulation of iron may play a role in enhancing oxidant-induced tubular injury by producing increased amounts of reactive oxygen species. From findings in cells from heme oxygenase-1 (HO-1)-deficient mice, HO-1 is postulated to prevent abnormal intracellular iron accumulation. Recently, it has been reported that HO-1 is induced in the renal tubular epithelial cells, in which iron is deposited after iron loading, and that this HO-1 induction may be involved in ameliorating iron-induced renal toxicity. We previously showed that chronic administration of angiotensin II to rats induces HO-1 expression in the tubular epithelial cells. These observations led us to investigate whether there is a link between iron deposition and HO-1 induction in renal tubular cells in rats undergoing angiotensin II infusion. In the present study, rats were given angiotensin II for continuously 7 days. Prussian blue staining revealed the distinct deposits of iron in the proximal tubular epithelial cells after angiotensin II administration. Electron microscopy demonstrated that iron particles were present in the lysosomes of these cells. Histologic and immunohistochemical analyses showed that stainable iron and immunoreactive ferritin and HO-1 were colocalized in the tubular epithelial cells. Treatment of angiotensin II-infused rats with an iron chelator, deferoxamine, blocked the abnormal iron deposition in kidneys and also the induced expression of HO-1 and ferritin expression. Furthermore, deferoxamine treatment suppressed the angiotensin II-induced increase in the urinary excretion of protein and N-acetyl-beta-D-glucosaminidase, a marker of tubular injury; however, deferoxamine did not affect the angiotensin II-induced decrease in glomerular filtration rate. These results suggest that angiotensin II causes renal injury, in part, by inducing the deposition of iron in the kidney.

Essential role(s) of the intrarenal renin-angiotensin system in transforming growth factor-beta1 gene expression and induction of hypertrophy of rat kidney proximal tubular cells in high glucose.

These studies investigated the question of whether the intrarenal renin-angiotensin system (RAS) is essential for transforming growth factor-beta1 (TGF-beta1) gene expression and induction of hypertrophy of renal proximal tubular cells in high glucose in vitro. Antisense and sense angiotensinogen (ANG) cDNAs were stably transfected into rat immortalized renal proximal tubular cells (IRPTC). ANG secretion from rat IRPTC was quantified by a specific RIA for rat ANG. Cellular ANG, TGF-beta1, and collagen alpha1 (type IV) mRNA levels were determined by Northern blot analysis or by reverse transcriptase-PCR assay. Hypertrophy of IRPTC was analyzed by Western blotting of cellular p27(Kip1) protein, flow cytometry, and cellular protein assay. The results showed that stable transfer of antisense ANG cDNA into IRPTC suppressed the basal TGF-beta1 and collagen alpha1 (type IV) mRNA expression and blocked the stimulatory effect of high glucose (i.e., 25 mM) on TGF-beta1 and collagen alpha1 (type IV) mRNA expression and induction of IRPTC hypertrophy. In contrast, stable transfer of sense ANG cDNA into IRPTC had no significant effect on these parameters. These data demonstrate that local intrarenal RAS activation is essential for TGF-beta1 gene expression and induction of hypertrophy of renal proximal tubular cells in high glucose.