Search PubMed⌕ Search

Biomedical subjects

J R Ingelfinger

Publications and source records attributed to J R Ingelfinger.

At least 19 recordsLinked to original sources
PubMed2006

Reactive oxygen species and nuclear factor-kappa B pathway mediate high glucose-induced Pax-2 gene expression in mouse embryonic mesenchymal epithelial cells and kidney explants.

Diabetic mellitus confers a major risk of congenital malformations, and is associated with diabetic embryopathy, affecting multiple organs including the kidney. The DNA paired box-2 (Pax-2) gene is essential in nephrogenesis. We investigated whether high glucose alters Pax-2 gene expression and aimed to delineate its underlying mechanism(s) of action using both in vitro (mouse embryonic mesenchymal epithelial cells (MK4) and ex vivo (kidney explant from Hoxb7-green florescent protein (GFP) mice) approaches. Pax-2 gene expression was determined by reverse transcriptase-polymerase chain reaction, Western blotting, and immunofluorescent staining. A fusion gene containing the full-length 5'-flanking region of the human Pax-2 promoter linked to a luciferase reporter gene, pGL-2/hPax-2, was transfected into MK4 cells with or without dominant negative IkappaBalpha (DN IkappaBalpha) cotransfection. Fusion gene expression level was quantified by cellular luciferase activity. Reactive oxygen species (ROS) generation was measured by lucigenin assay. Embryonic kidneys from Hoxb7-GFP mice were cultured ex vivo. High D(+) glucose (25 mM), compared to normal glucose (5 mM), specifically induced Pax-2 gene expression in MK4 cells and kidney explants. High glucose-induced Pax-2 gene expression is mediated, at least in part, via ROS generation and activation of the nuclear factor kappa B signaling pathway, but not via protein kinase C, p38 mitogen-activated protein kinase (MAPK), and p44/42 MAPK signaling.

Animals↗
PubMed2006

Albumin induces endoplasmic reticulum stress and apoptosis in renal proximal tubular cells.

Chronic proteinuria appears to be a key factor in tubulointerstitial damage. Recent studies have emphasized a pathogenic role of endoplasmic reticulum (ER) stress which is induced by the accumulation of misfolded proteins in ER, extracellular stress, etc. In the present study, we investigated ER stress and ER stress-induced apoptosis in proximal tubular cells (PTCs). Immortalized rat PTCs (IRPTCs) were cultured with bovine serum albumin (BSA). The viability of IRPTCs decreased proportionately with BSA overload in a time-dependent manner. Quantitative real-time polymerase chain reaction analysis revealed that 40 mg/ml BSA increases mRNA of ER stress markers by 7.7- and 4.6-fold (glucose-regulated protein 78 (GRP78) and oxygen-regulated protein 150 (ORP150), respectively) as compared to control. The increased expression of ORP150 and GRP78 in IRPTCs with albumin overload was detected by Western blot and immunofluorescence study. These in vitro observations were supported by in vivo studies, which demonstrated that ER stress proteins were upregulated at PTCs in experimental proteinuric rats. Furthermore, increased ER stress-induced apoptosis and activation of caspase-12 were observed in IRPTCs with albumin overload and kidneys of experimental proteinuric rats. We confirmed that apoptotic cell death was attenuated by co-incubation with caspase-3 inhibitor or calpain inhibitors. These results indicate that the ER stress-induced apoptosis pathway contributed to the insult of tubular cells by proteinuria. In conclusion, renal tubular cells exposed to high protein load suffer from ER stress. ER stress may subsequently lead to tubular damage by activation of caspase-12.

Acrylates↗
PubMed2006

Upregulation of osteopontin gene expression in diabetic rat proximal tubular cells revealed by microarray profiling.

Progression of diabetic nephropathy appears directly related to renal tubulointerstitial injury, but the involved genes are incompletely delineated. To identify such genes, DNA microarray analysis was performed with RNA from renal proximal tubules (RPTs) of streptozotocin-induced diabetic Wistar rats, spontaneously diabetic BioBreeding rats, and rat immortalized renal proximal tubular cells (IRPTCs) exposed to high glucose (25 mM) medium for 2 weeks. Osteopontin (OPN) mRNA expression was quantified by real time-quantitative polymerase chain reaction (RT-qPCR) or conventional reverse transcriptase-polymerase chain reaction (RT-PCR). OPN mRNA expression was upregulated (5-70-fold increase) in diabetic rat RPTs and in IRPTCs chronically exposed to high glucose compared to control RPTs and IRPTCs. High glucose, angiotensin II, phorbol 12-myristate 13-acetate and transforming growth factor-beta 1 (TGF-beta1) stimulated OPN mRNA expression in IRPTCs in a dose- and time-dependent manner. This effect was inhibited by tiron, taurine, diphenylene iodinium, losartan, perindopril, calphostin C, or LY 379196 but not PD123319. IRPTCs overexpressing dominant-negative protein kinase C-beta 1 (PKC-beta1) cDNA or antisense TGF-beta1 cDNA prevented the high glucose effect on OPN mRNA expression. We concluded that high glucose-mediated increases in OPN gene expression in diabetic rat RPTs and IRPTCs are mediated, at least in part, via reactive oxygen species generation, intrarenal rennin-angiotensin system activation, TGF-beta1 expression, and PKC-beta1 signaling.

Angiotensin II↗
PubMed2006

RAS blockade decreases blood pressure and proteinuria in transgenic mice overexpressing rat angiotensinogen gene in the kidney.

Angiotensinogen (ANG) is the sole substrate of the renin-angiotensin system (RAS). Clinical studies have shown that RAS activation may lead to hypertension, a major cardiovascular and renal risk factor. To delineate the underlying mechanisms of hypertension-induced nephropathy, we generated transgenic mice that overexpress rat ANG (rANG) in the kidney to establish whether intrarenal RAS activation alone can evoke hypertension and kidney damage and whether RAS blockade can reverse these effects. Transgenic mice overexpressing renal rANG were generated by employing the kidney-specific, androgen-regulated protein promoter linked to rANG cDNA. This promoter targets rANG cDNA to renal proximal tubules and responds to androgen stimulation. Transgenic mice displayed kidney-specific expression of rANG, significantly increased blood pressure (BP) and albuminuria in comparison to non-transgenic littermates. Administration of losartan (an angiotensin II (type 1)-receptor antagonist) or perindopril (an angiotensin-converting enzyme inhibitor) reversed these abnormalities in transgenic animals. Renal injury was evident on examination of the kidneys in transgenic mice, and attenuated by losartan and perindopril treatment. We conclude that the overproduction of ANG alone in the kidney induces an increase in systemic BP, proteinuria, and renal injury. RAS blockers prevent these abnormalities. These data support the role of the intrarenal RAS in the development of hypertension and renal injury.

Angiotensin II Type 1 Receptor Blockers↗
PubMed2006

Transforming growth factor-beta 1 stimulates angiotensinogen gene expression in kidney proximal tubular cells.

The present study investigated whether transforming growth factor-beta 1 (TGF-beta1) exerts an autocrine positive effect on angiotensinogen (ANG) gene expression in rat kidney proximal tubular cells, and delineates its underlying mechanism(s) of action. Rat immortalized renal proximal tubular cells (IRPTCs) and freshly isolated mouse renal proximal tubules were incubated in the absence or presence of active human TGF-beta1. IRPTCs were also stably transfected with rat TGF-beta1 or p53 tumor suppressor protein (p53) cDNA in sense (S) and antisense (AS) orientations. ANG mRNA and p53 protein expression were assessed by reverse transcription-polymerase chain reaction and Western blotting, respectively. Reactive oxygen species (ROS) generation was quantified by lucigenin assay. Active TGF-beta1 evoked ROS generation and stimulated ANG mRNA and p53 protein expression, whereas a superoxide scavenger and inhibitors of nicotinamide adenine dinucleotide oxidase and p38 mitogen-activated protein kinase (p38 MAPK) abolished the TGF-beta1 effect. Stable transfer of p53 cDNA (S) enhanced and p53 cDNA (AS) abolished the stimulatory effect of TGF-beta1 on ANG mRNA expression in IRPTCs. Our results demonstrate that TGF-beta1 stimulates ANG gene expression and its action is mediated, at least in part, via ROS generation, p38 MAPK activation, and p53 expression, suggesting that angiotensin II and TGF-beta1 may form a positive feedback loop to enhance their respective gene expression, leading to renal injury.

Angiotensinogen↗
PubMed2005

Shiga toxin enhances functional tissue factor on human glomerular endothelial cells: implications for the pathophysiology of hemolytic uremic syndrome.

BACKGROUND: The pathogenesis of Shiga toxin (Stx)-mediated childhood hemolytic uremic syndrome (HUS) is not fully delineated, although current evidence implicates a prothrombotic state. We hypothesized that the tissue factor (TF) pathway plays a major role in the pathophysiology of HUS. MATERIALS AND METHODS: We measured cell surface TF activity in response to tumor necrosis factor-alpha (TNF-alpha) (20 ng mL(-1), 2-144 h), Stx-1 (10(-11) mol L(-1), 4-144 h), or their combination (TNF-alpha 22 h and Stx-1 for the last 0.5-4 h of TNF-alpha incubation) on human glomerular (microvascular) endothelial cells (HGECs) and human umbilical vein (macrovascular) endothelial cells (HUVECs). RESULTS AND CONCLUSIONS: We observed that while TNF-alpha caused an increase in cell surface TF activity on both cell types, the combination of TNF-alpha and Stx-1 differentially affected HGECs. On these cells, TF activity was increased further by 2.67 +/- 0.38-fold (n = 38, P < 0.001), consistent with our parallel observation that Stx-1 binds to HGECs but not to HUVECs. Anti-TF antibody abolished functional TF while anti-tissue factor pathway inhibitor antibody enhanced TF activity. Stx-1 alone did not induce TF activity on either cell type. Measurement of TF antigen levels and quantitative real-time polymerase chain reaction demonstrated that exposure to TNF-alpha markedly increased TF protein and TF mRNA for HGECs, but the exposure to the combination of TNF-alpha and Stx-1 did not increase further the amount of either TF protein or TF mRNA. We conclude that cytokine-activated HGECs, but not HUVECs, undergo a significant augmentation of cell surface TF activity following exposure to Stx, suggesting an important role for TF in the coagulopathy observed in HUS.

Cell Membrane↗
PubMed2002

Hyperglycemia induces insulin resistance on angiotensinogen gene expression in diabetic rat kidney proximal tubular cells.

Clinical and animal studies have shown that treatment with angiotensin-converting enzyme (ACE) inhibitors or angiotensin II (Ang II) receptor antagonists slows the progression of nephropathy in diabetes, indicating that Ang II plays an important role in its development. We have reported previously that insulin inhibits the stimulatory effect of high glucose levels on angiotensinogen (ANG) gene expression in rat immortalized renal proximal tubular cells (IRPTCs) via the mitogen-activated protein kinase (p44/42 MAPK) signal transduction pathway. We hypothesize that the suppressive action of insulin on ANG gene expression might be attenuated in renal proximal tubular cells (RPTCs) of rats with established diabetes. Two groups of male adult Wistar rats were studied: controls and streptozotocin (STZ)-induced diabetic rats at 2, 4, 8 and 12 weeks post-STZ administration. Kidney proximal tubules were isolated and cultured in either normal glucose (i.e. 5 mM) or high glucose (i.e. 25 mM) medium to determine the inhibitory effect of insulin on ANG gene expression. Immunoreactive rat ANG (IR-rANG) in culture media and cellular ANG mRNA were measured by a specific radioimmunoassay and reverse transcription-polymerase chain reaction assay respectively. Activation of the p44/42 MAPK signal transduction pathway in rat RPTCs was evaluated by p44/42 MAPK phosphorylation employing a PhosphoPlus p44/42 MAPK antibody kit. Insulin (10(-7) M) inhibited the stimulatory effect of high glucose levels on IR-rANG secretion and ANG gene expression and increased p44/42 MAPK phosphorylation in normal rat RPTCs. In contrast, it failed to affect these parameters in diabetic rat RPTCs. In conclusion, our studies demonstrate that hyperglycaemia induces insulin resistance on ANG gene expression in diabetic rat RPTCs by altering the MAPK signal transduction pathway.

Angiotensinogen↗
PubMed2001

Maternal protein restriction suppresses the newborn renin-angiotensin system and programs adult hypertension in rats.

Restriction of maternal protein intake during rat pregnancy produces offspring that are hypertensive in adulthood, but the mechanisms are not well understood. Our purpose was to determine whether this adult hypertension could be programmed during development by suppression of the fetal/newborn renin-angiotensin system (RAS) and a consequent reduction in nephron number. Pregnant rats were fed a normal protein (19%, NP) or low-protein (8.5%, LP) diet throughout gestation. Birth weight was reduced by 13% (p < 0.0005), and the kidney/body weight ratio was reduced in LP pups. Renal renin mRNA levels were significantly reduced in newborn LP pups; renal renin concentration and renin immunostaining were suppressed. Renal tissue angiotensin II levels were also suppressed in newborn LP (0.079 +/- 0.002 ng/mg, LP versus 0.146 +/- 0.016 ng/mg, NP, p < 0.01). Mean arterial pressure in conscious, chronically instrumented adult offspring (21 wk) was higher in LP (135 +/- 1 mm Hg, LP versus 126 +/- 1 mm Hg, NP, p < 0.00007), and GFR normalized to kidney weight was reduced in LP (p < 0.04). The number of glomeruli per kidney was lower in adult LP offspring (21,567 +/- 1,694, LP versus 28,917 +/- 2,342, NP, p < 0.03), and individual glomerular volume was higher (1.81 +/- 0.16 10(6) microm(3), LP versus 1.11 +/- 0.10 10(6) microm(3), NP, p < 0.005); the total volume of all glomeruli per kidney was not significantly different. Thus, perinatal protein restriction in the rat suppresses the newborn intrarenal RAS and leads to a reduced number of glomeruli, glomerular enlargement, and hypertension in the adult.

Animals↗
PubMed2001

Characterization of a putative insulin-responsive element and its binding protein(s) in rat angiotensinogen gene promoter: regulation by glucose and insulin.

We previously demonstrated that high glucose activates angiotensinogen (ANG) expression and that insulin inhibits this activation. The present studies aim to investigate whether insulin regulates ANG gene expression in kidney proximal tubular cells at the transcription level via interaction of the putative insulin-response element (IRE) with its binding protein(s) in the 5'-flanking region of the ANG gene. Fusion genes containing various lengths of the 5'-flanking region of the rat ANG gene fused to a human GH (hGH) gene as reporter were constructed and transiently introduced into rat immortalized renal proximal tubular cells (IRPTCs). The expression of the fusion genes was monitored by the amount of immunoreactive hGH secreted into the medium as assayed by a specific RIA for hGH. Insulin inhibited the expression of pOGH (rANG N-1498/+18), pOGH (rANG N-1120/+18) and pOGH (rANG N-882/+18) but not pOGH (rANG N-854/+18), pOGH (rANG N-820/+18), pOGH (rANG N-688/+18) and pOGH (rANG N-53/+18) in high-glucose (i.e. 25 mM) medium. Site-directed mutagenesis of nucleotides N-874 to N-867 (5' CCC GCC CT 3') in the 5'-flanking region of the rat ANG gene abolished the response to insulin. Insulin also inhibited the expression of the fusion gene containing the DNA fragment ANG N-882 to N-855 inserted upstream of the ANG gene promoter (N-53/+18), but had no effect on a mutant of N-882 to N-855. Gel mobility shift assays revealed that the labeled putative rat ANG-IRE motif (N-878 to N-864, 5' CCT TCC CGC CCT TCA 3') was bound to the nuclear proteins of IRPTCS: This binding was displaced by unlabeled ANG-IRE and IRE of human glyceraldehyde phosphate dehydrogenase but not by mutants of ANG-IRE and IRE of the rat glucagon gene. Southwestern blotting analysis revealed that the labeled putative ANG-IRE motif bound to a major nuclear protein with an apparent molecular mass of 48 kDA: Finally, high glucose levels enhanced 48-kDa nuclear protein expression and induced an additional 70-kDa nuclear protein expression in IRPTCs, as revealed by Southwestern blotting. Insulin inhibited both 48- and 70-kDa nuclear proteins expression induced by high glucose levels. Its inhibitory effect was reversed by the presence of PD98059 (an inhibitor of mitogen-activated protein kinase, MAPK) but not by wortmannin (an inhibitor of phosphatidylinositol 3- kinase). These studies demonstrate that insulin action on ANG gene expression is at the transcriptional level. The molecular mechanism (s) of insulin action is mediated, at least in part, via interaction of the functional IRE with unidentified 48- and 70- kDa nuclear proteins in the rat ANG gene and is MAPK dependent.

Angiotensinogen↗
PubMed2000

Heme oxygenase-1 is upregulated in the kidney of angiotensin II-induced hypertensive rats : possible role in renoprotection.

In this study, we investigated the regulation and physiological role of heme oxygenase-1 (HO-1) in the kidney of rats with hypertension. Rats were continuously administered either angiotensin II (Ang II) or norepinephrine with an osmotic minipump for up to 7 days. Ang II infusion decreased the glomerular filtration rate (GFR) as determined through creatinine clearance (3.2+/-0.2 versus 1.2+/-0.2 mL/min with Ang II infusion, P<0.01) and increased proteinuria (9. 7+/-1.3 versus 28.1+/-7.2 mg/d with Ang II infusion, P<0.01). In contrast, norepinephrine did not alter these laboratory values. Ang II infusion significantly increased HO-1 expression in mRNA (442+/-98% of control at day 5, P<0.01) and protein levels (314+/-49% of control at day 5, P<0.01). Immunohistochemistry showed that in the kidney of normotensive rats, HO-1 was expressed mainly in the basal side in the renal tubules. After Ang II infusion, HO-1 staining was more extensively dispersed in the tubular epithelial cells. The intraperitoneal administration of zinc protoporphyrin, an HO inhibitor, to Ang II-infused rats further decreased GFR (0.8+/-0. 1 mL/min) and increased proteinuria (52.5+/-13.0 mg/d). In contrast, the administration of hemin, an HO inducer, ameliorated the Ang II-induced decrease in GFR (2.4+/-0.2 mL/min) and increase in proteinuria (9.3+/-4.5 mg/d). These data suggest that HO-1 upregulation in the kidney of Ang II-induced hypertensive rats may exert a renoprotective effect against Ang II-induced renal injury.

Angiotensin II↗
PubMed2000

High levels of glucose stimulate angiotensinogen gene expression via the P38 mitogen-activated protein kinase pathway in rat kidney proximal tubular cells.

The present studies investigated whether the effect of high levels of glucose on angiotensinogen (ANG) secretion and gene expression in kidney proximal tubular cells is mediated at least in part via the activation of p38 mitogen-activated protein kinase (p38 MAPK). Rat immortalized renal proximal tubular cells (IRPTCs) were cultured in monolayer. The levels of immunoreactive rat ANG (IR-rANG) secreted into the medium and the levels of cellular ANG messenger RNA were determined by a specific RIA for rat ANG and a RT-PCR assay, respectively. Phosphorylation of cellular p38 MAPK was determined by Western blot analysis using the Phospho Plus p38 MAPK antibody kit. High levels of glucose (i.e. 25 mM) and phorbol 12-myristate 13-acetate (PMA; 10(-7) M) increased the secretion of IR-rANG and cellular ANG messenger RNA as well as phosphorylation of p38 MAPK in IRPTCs. This stimulatory effect of high levels of glucose and PMA was blocked by SB 203580 (a specific inhibitor of p38 MAPK), but not by SB 202474 (a negative control of SB 203580). High levels of D-sorbitol or 2-deoxy-D-glucose (i.e. > or = 35 mM) also stimulated the phosphorylation of p38 MAPK, but did not stimulate ANG secretion or gene expression. GF 109203X (an inhibitor of protein kinase C) blocked the stimulatory effect of high levels of glucose and PMA on ANG gene expression, whereas it did not block the effect of high levels of glucose, sorbitol, or 2-deoxy-D-glucose on p38 MAPK phosphorylation in IRPTCs. These studies demonstrate that the stimulatory effect of a high level of glucose (25 mM) on ANG gene expression in IRPTCS may be mediated at least in part via activation of p38 MAPK signal transduction pathway and is protein kinase C independent.

Angiotensinogen↗
PubMed1999

Postnatal transient renal insufficiency in the feto-fetal transfusion syndrome.

Twinning and higher-order multiple-gestation pregnancies have become relatively frequent in the current era of assisted reproductive techniques. Vascular interconnections are present in nearly all monochorionic twin placentae, yet hemodynamically significant arteriovenous anastomoses resulting in the feto-fetal transfusion syndrome occur in only 5%-18% of these. When arteriovenous connections through a shared placental cotyledon are present, variable amounts of blood may be transfused from one fetus to the other, and feto-fetal transfusion syndrome may result. While reports of renal failure due to a small non-functioning kidney in the donor infant pre- or postnatally have been published, recoverable renal insufficiency has not been previously delineated in feto-fetal transfusion syndrome. This article describes a case of postnatal transient renal insufficiency in a donor infant from a pair of monozygotic twins.

Acute Kidney Injury↗
PubMed1999

Molecular mechanisms of glucose action on angiotensinogen gene expression in rat proximal tubular cells.

BACKGROUND: Clinical studies have shown that the angiotensin-converting enzyme (ACE) inhibitors or angiotensin II (Ang II) receptor antagonists decrease proteinuria and slow the progression of nephropathy in diabetes, indicating that Ang II plays an important role in the development of nephropathy. We have previously reported that high levels of glucose stimulate the expression of rat angiotensinogen (ANG) gene in opossum kidney (OK) proximal tubular cells. We hypothesized that the stimulatory effect of D(+)-glucose on the expression of the ANG gene in kidney proximal tubular cells is mediated via de novo synthesis of diacylglycerol (DAG) and the protein kinase C (PKC) signal transduction pathway. METHODS: Immortalized rat proximal tubular cells (IRPTCs) were cultured in monolayer. The stimulatory effect of glucose on the activation of polyol pathway and PKC signal transduction pathway in IRPTCs was determined. The immunoreactive rat ANG (IR-rANG) in the culture medium and the cellular ANG mRNA were measured with a specific radioimmunoassay and a reverse transcription-polymerase chain reaction assay, respectively. RESULTS: D(+)-glucose (25 mM) markedly increased the intracellular levels of sorbitol, fructose, DAG, and PKC activity as well as the expression of IR-rANG and ANG mRNA in IRPTCs. These stimulatory effects of D(+)-glucose (25 mM) were blocked by an inhibitor of aldose reductase, Tolrestat. PKC inhibitors also inhibited the stimulatory effect of D(+)-glucose (25 mM) on the expression of the IR-rANG in IRPTCs. The addition of phorbol 12-myristate 13-acetate further enhanced the stimulatory effect of D(+)-glucose (25 mM) on the expression of the IR-rANG in IRPTCs and blocked the inhibitory effect of Tolrestat. CONCLUSION: These studies suggest that the stimulatory effect of a high level of D(+)-glucose (25 mM) on the expression of the ANG gene in IRPTCs is mediated, at least in part, via the de novo synthesis of DAG, an activator of PKC signal transduction pathway.

Aldehyde Reductase↗
PubMed1999

Glucocorticoid metabolism in proximal tubules modulates angiotensin II-induced electrolyte transport.

The hormonal interactions that regulate electrolyte transport in the proximal tubule are complex and incompletely understood. Since endogenous glucocorticoids and angiotensin II each can affect electrolyte transport in this renal segment, we hypothesized that local metabolism of glucocorticoids by the enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) might alter the response to angiotensin II. Studies were conducted in cultured origin defective SV-40 transformed immortalized renal proximal tubule cells (IRPTC) derived from weanling Wistar rat kidney. The 11beta-HSD contained in these cells uses NADP+, has an apparent Km for corticosterone of 1.6 microM, but functions only as a dehydrogenase (corticosterone --> 11-dehydro-corticosterone). When mounted in modified Ussing chambers, IRPTC generate a transmembrane current, and angiotensin II (10 pM to 10 microM) increases this sodium-dependent current. Cells incubated with corticosterone (100 nM) and the 11beta-HSD inhibitor carbenoxolone (CBX) (1 microM) for 24 hr and then acutely stimulated with angiotensin (10 nM) show a greater rise in current than do cells exposed to corticosterone alone and stimulated with angiotensin (corticosterone + CBX: 64.2% +/- 20.5% vs. corticosterone: 18.8% +/- 5.9%; P < 0.02 at 180 min)[mean +/- SE percentage above baseline, n = 8/group]. Cells exposed to corticosterone (100 nM) or CBX (1 microM) alone for 24 hr and then stimulated with angiotensin II (10 nM) had responses similar to controls. Thus glucocorticoids can enhance angiotensin II-induced electrolyte transport in proximal tubule epithelial cells when local 11beta-HSD is inhibited.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1999

Rat proximal tubule cell line transformed with origin-defective SV40 DNA: autocrine ANG II feedback.

The renal proximal tubule (PT) is a major site for a complete tissue renin-angiotensin system (RAS) and produces endogenous angiotensin II (ANG II). The present studies demonstrate autocrine RAS feedback in a line of origin-defective SV40 plasmid transformed immortalized rat PT cells (IRPTC) designated as line 93-p-2-1, which are highly differentiated and express all RAS components. Receptor competition assays and Southern blot following RT-PCR demonstrated that these IRPTC express AT1 and AT2 angiotensin receptor subtypes. Autocrine RAS feedback was examined following exposure to ANG II (10(-8) M), and it was noted that angiotensinogen mRNA increases significantly by 1 h and remains elevated through 24 h. The AT1 blocker losartan prevents this increase. Moreover, ANG II upregulates expression of ANG II receptor mRNA (both AT1 and AT2). Thus the present studies demonstrate positive ANG II feedback with angiotensinogen and ANG II receptors in PTC, suggesting that the main site of such intrarenal feedback in vivo is within PT. ANG II secreted by line 93-p-2-1 is increased by isoproterenol, suggesting beta-adrenergic regulation in IRPTC.

Angiotensin II↗
PubMed1999

Comparison of tissue factor pathway in human umbilical vein and adult saphenous vein endothelial cells: implications for newborn hemostasis and for laboratory models of endothelial cell function.

In this work we have undertaken a comparative study of human umbilical vein endothelial cells (HUVECs) and human saphenous vein endothelial cells (HSVECs) with respect to functional and antigenic tissue factor (TF), tissue factor pathway inhibitor (TFPI), and TF mRNA. Monolayers of each cell type (passage 2, except where specified) were grown to confluence and then activated for 4 h with either 50 U/mL IL-1-alpha or 10 microg/mL tumor necrosis factor-alpha. Activated factor X appearing in supernatant was measured using a chromogenic assay, and both Northern blots and quantitative RT-PCR were performed to assess concentrations of TF mRNA accompanying activation. The role of TFPI was separately determined by ELISA for supernatant TFPI antigen, and by measurements of production of activated factor X in the presence of 0, 5, 15, or 50 microg/mL of an antibody directed against TFPI. To address a non-TF pathway endothelial cell function, antigenic concentrations of tissue plasminogen activator for both cell types was also determined by ELISA. HUVECs were found to produce 2.4- to 3.5-fold more functional TF. No significant HUVEC-HSVEC differences were detected in TF antigen, supernatant TFPI, anti-TFPI affinity for endothelial cell-associated TFPI, TF mRNA or its amplification products, and tissue plasminogen activator. Immunostaining for TF antigen, however, may have failed to detect a modest HUVEC-HSVEC difference. Our finding with respect to functional TF indicates that HUVECs and HSVECs are not equivalent in terms of models for endothelial cell function in small children versus adults.

Adult↗
PubMed1999

Insulin inhibits angiotensinogen gene expression via the mitogen-activated protein kinase pathway in rat kidney proximal tubular cells.

The present study aimed to investigate the molecular mechanism(s) of insulin action on angiotensinogen (ANG) secretion and gene expression in kidney proximal tubular cells exposed to high levels of glucose. Immortalized rat proximal tubular cells (IRPTC) were cultured in monolayer. The levels of rat ANG and ANG messenger RNA in the IRPTC were quantified by a specific RIA for rat ANG (RIA-rANG) and by an RT-PCR assay. Insulin inhibited the stimulatory effect of a high level of glucose (25 mM) and phorbol 12-myristate 13-acetate, an activator of protein kinase C) on the secretion of ANG and the expression of the ANG messenger RNA in IRPTC. This inhibitory action of insulin on the ANG secretion and gene expression was blocked by PD98059 (an inhibitor of mitogen-activated protein kinase kinase) but not by Wortmannin (an inhibitor of phosphatidylinositol-3-kinase). PD98059 was effective in inhibiting the phosphorylation of MEK 1/2 and p44/42 MAP kinase in IRPTC stimulated by insulin. These studies demonstrate that insulin prevents the stimulatory effect of high levels of glucose on the expression of the renal ANG gene in IRPTC, at least in part, via the MAPK kinase signal transduction pathway, subsequently inhibiting the activation of the local renal renin-angiotensin system.

Androstadienes↗