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1.
PLoS One ; 13(8): e0201293, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30067804

RESUMO

Fructose consumption causes metabolic diseases and renal injury primarily in the renal cortex where fructose is metabolized. Analyzing gene differential expression induced by dietary manipulation is challenging. The effects may depend on the base diet and primary changes likely induce secondary or higher order changes that are difficult to capture by conventional univariate transcriptome analyses. We hypothesized that dietary fructose induces a genetic program in the kidney cortex that favors lipogenesis and gluconeogenesis. To test this, we analyzed renal cortical transcriptomes of rats on normal- and high-salt base diets supplemented with fructose. Both sets of data were analyzed using the Characteristic Direction method to yield fructose-induced gene vectors of associated differential expression values. A fructose-specific "signature" of 139 genes differentially expressed was extracted from the 2 diet vectors by a new algorithm that takes into account a gene's rank and standard deviation of its differential expression value. Of these genes, 97 were annotated and the top 34 accounted for 80% of the signal in the annotated signature. The genes were predominantly proximal tubule-specific, coding for metabolic enzymes or transporters. Cosine similarity of signature genes in the two fructose-induced vectors was >0.78. These 139 genes of the fructose signature contributed 27% and 38% of total differential expression on normal- and high- salt diet, respectively. Principal Component Analysis showed that the individual animals could be grouped according to diet. The fructose signature contained a greater enrichment of Gene Ontology processes related to nutrition and metabolism of fructose than two univariate analysis methods. The major feature of the fructose signature is a change in metabolic programs of the renal proximal tubule consistent with gluconeogenesis and de-novo lipogenesis. This new "signature" constitutes a new metric to bridge the gap between physiological phenomena and differential expression profile.


Assuntos
Frutose/metabolismo , Córtex Renal/metabolismo , Transcriptoma , Animais , Dieta , Frutose/genética , Regulação da Expressão Gênica , Gluconeogênese , Nefropatias/genética , Nefropatias/metabolismo , Lipogênese , Masculino , Doenças Metabólicas/genética , Doenças Metabólicas/metabolismo , Ratos , Ratos Sprague-Dawley
3.
Free Radic Biol Med ; 84: 322-330, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25862412

RESUMO

Excess circulating iron is stored in the liver, and requires reduction of non-Tf-bound iron (NTBI) and transferrin (Tf) iron at the plasma membrane and endosomes, respectively, by ferrireductase (FR) proteins for transport across biological membranes through divalent metal transporters. Here, we report that prion protein (PrP(C)), a ubiquitously expressed glycoprotein most abundant on neuronal cells, functions as a FR partner for divalent-metal transporter-1 (DMT1) and ZIP14. Thus, absence of PrP(C) in PrP-knock-out (PrP(-/-)) mice resulted in markedly reduced liver iron stores, a deficiency that was not corrected by chronic or acute administration of iron by the oral or intraperitoneal routes. Likewise, preferential radiolabeling of circulating NTBI with (59)Fe revealed significantly reduced uptake and storage of NTBI by the liver of PrP(-/-) mice relative to matched PrP(+/+) controls. However, uptake, storage, and utilization of ferritin-bound iron that does not require reduction for uptake were increased in PrP(-/-) mice, indicating a compensatory response to the iron deficiency. Expression of exogenous PrP(C) in HepG2 cells increased uptake and storage of ferric iron (Fe(3+)), not ferrous iron (Fe(2+)), from the medium, supporting the function of PrP(C) as a plasma membrane FR. Coexpression of PrP(C) with ZIP14 and DMT1 in HepG2 cells increased uptake of Fe(3+) significantly, and surprisingly, increased the ratio of N-terminally truncated PrP(C) forms lacking the FR domain relative to full-length PrP(C). Together, these observations indicate that PrP(C) promotes, and possibly regulates, the uptake of NTBI through DMT1 and Zip14 via its FR activity. Implications of these observations for neuronal iron homeostasis under physiological and pathological conditions are discussed.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , FMN Redutase/metabolismo , Proteínas PrPC/fisiologia , Animais , Transporte Biológico , Células Hep G2 , Humanos , Ferro/metabolismo , Fígado/metabolismo , Camundongos Knockout
4.
J Biol Chem ; 290(9): 5512-22, 2015 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-25572394

RESUMO

Brain iron-dyshomeostasis is an important cause of neurotoxicity in prion disorders, a group of neurodegenerative conditions associated with the conversion of prion protein (PrP(C)) from its normal conformation to an aggregated, PrP-scrapie (PrP(Sc)) isoform. Alteration of iron homeostasis is believed to result from impaired function of PrP(C) in neuronal iron uptake via its ferrireductase activity. However, unequivocal evidence supporting the ferrireductase activity of PrP(C) is lacking. Kidney provides a relevant model for this evaluation because PrP(C) is expressed in the kidney, and ∼370 µg of iron are reabsorbed daily from the glomerular filtrate by kidney proximal tubule cells (PT), requiring ferrireductase activity. Here, we report that PrP(C) promotes the uptake of transferrin (Tf) and non-Tf-bound iron (NTBI) by the kidney in vivo and mainly NTBI by PT cells in vitro. Thus, uptake of (59)Fe administered by gastric gavage, intravenously, or intraperitoneally was significantly lower in PrP-knock-out (PrP(-/-)) mouse kidney relative to PrP(+/+) controls. Selective in vivo radiolabeling of plasma NTBI with (59)Fe revealed similar results. Expression of exogenous PrP(C) in immortalized PT cells showed localization on the plasma membrane and intracellular vesicles and increased transepithelial transport of (59)Fe-NTBI and to a smaller extent (59)Fe-Tf from the apical to the basolateral domain. Notably, the ferrireductase-deficient mutant of PrP (PrP(Δ51-89)) lacked this activity. Furthermore, excess NTBI and hemin caused aggregation of PrP(C) to a detergent-insoluble form, limiting iron uptake. Together, these observations suggest that PrP(C) promotes retrieval of iron from the glomerular filtrate via its ferrireductase activity and modulates kidney iron metabolism.


Assuntos
FMN Redutase/metabolismo , Ferro/metabolismo , Rim/metabolismo , Proteínas PrPC/metabolismo , Animais , Western Blotting , Linhagem Celular Transformada , Membrana Celular/metabolismo , FMN Redutase/genética , Feminino , Transporte de Íons/genética , Ferro/farmacocinética , Radioisótopos de Ferro , Rim/citologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Masculino , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Proteínas PrPC/genética , Transferrina/metabolismo , Transferrina/farmacocinética
5.
Science ; 345(6201): 1130, 2014 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-25190787

RESUMO

Glykys et al. (Reports, 7 February 2014, p. 670) conclude that, rather than ion transporters, "local impermeant anions establish the neuronal chloride concentration" and thereby determine "the magnitude and direction of GABAAR currents at individual synapses." If this were possible, perpetual ion-motion machines could be constructed. The authors' conclusions conflict with basic thermodynamic principles.


Assuntos
Encéfalo/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Neurônios/metabolismo , Receptores de GABA-A/metabolismo , Animais
6.
Kidney Int ; 85(3): 561-9, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24132210

RESUMO

Renal proximal tubule cells from spontaneously hypertensive rats (SHR), compared with normotensive Wistar-Kyoto rats (WKY), have increased oxidative stress. The contribution of mitochondrial oxidative phosphorylation to the subsequent hypertensive phenotype remains unclear. We found that renal proximal tubule cells from SHR, relative to WKY, had significantly higher basal oxygen consumption rates, adenosine triphosphate synthesis-linked oxygen consumption rates, and maximum and reserve respiration. These bioenergetic parameters indicated increased mitochondrial function in renal proximal tubule cells from SHR compared with WKY. Pyruvate dehydrogenase complex activity was consistently higher in both renal proximal tubule cells and cortical homogenates from SHR than those from WKY. Treatment for 6 days with dichloroacetate, an inhibitor of pyruvate dehydrogenase kinase, significantly increased renal pyruvate dehydrogenase complex activity and systolic blood pressure in 3-week-old WKY and SHR. Therefore, mitochondrial oxidative phosphorylation is higher in renal proximal tubule cells from SHR compared with WKY. Thus, the pyruvate dehydrogenase complex is a determinant of increased mitochondrial metabolism that could be a causal contributor to the hypertension in SHR.


Assuntos
Hipertensão/metabolismo , Túbulos Renais Proximais/metabolismo , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Pressão Sanguínea , Células Cultivadas , Glicólise , Túbulos Renais Proximais/citologia , Masculino , Complexo Piruvato Desidrogenase/metabolismo , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY
8.
J Hypertens ; 30(6): 1176-84, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22504846

RESUMO

BACKGROUND: The renin-angiotensin (Ang) system controls blood pressure, in part, by regulating renal tubular sodium transport. In the kidney, activation of the angiotensin II type 1 (AT(1)) receptor increases renal sodium reabsorption, whereas the angiotensin II type 2 (AT(2)) receptor produces the opposite effect. We hypothesized that the AT(2) receptor regulates AT(1) receptor expression and function in the kidney. METHODS AND RESULTS: In immortalized renal proximal tubule (RPT) cells from Wistar-Kyoto rats, CGP42112, an AT(2) receptor agonist, decreased AT(1) receptor mRNA and protein expression (P < 0.05), as assessed by reverse transcriptase-polymerase chain reaction and immunoblotting. The inhibitory effect of the AT(2) receptor on AT(1) receptor expression was blocked by the AT(2) receptor antagonist, PD123319 (10 (-6)mol/l), the nitric oxide synthase inhibitor N(w)-nitro-L-arginine methyl ester (10(-4) mol/l), or the nitric oxide-dependent soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one (10(-5) mol/l), indicating that both nitric oxide and cyclic guanosine monophosphate (cGMP) were involved in the signaling pathway. Furthermore, CGP42112 decreased Sp1 serine phosphorylation and reduced the binding of Sp1 to AT(1) receptor DNA. Stimulation with Ang II (10(-11) mol/l per 30 min) enhanced Na(+)-K(+)-ATPase activity in RPT cells, which was prevented by pretreatment with CGP42112 (10(-7) mol/l per 24 h) (P < 0.05). The above-mentioned results were confirmed in RPT cells from AT(2) receptor knockout mice; AT(1) receptor expression and Ang II-stimulated Na-K-ATPase activity were greater in these cells than in RPT cells from wild-type mice (P < 0.05). AT(1)/AT(2) receptors co-localized and co-immunoprecipitated in RPT cells; short-term CGP42112 (10 mol/l per 30 min) treatment increased AT(1)/AT(2) receptor co-immunoprecipitation (P < 0.05). CONCLUSIONS: These results indicate that the renal AT(2) receptor, via nitric oxide/cGMP/Sp1 pathway, regulates AT(1 )receptor expression and function, which may be important in the regulation of sodium excretion and blood pressure.


Assuntos
GMP Cíclico/metabolismo , Túbulos Renais Proximais/metabolismo , Óxido Nítrico/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Fator de Transcrição Sp1/metabolismo , Animais , Sequência de Bases , Linhagem Celular Transformada , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Túbulos Renais Proximais/enzimologia , Microscopia Confocal , Oligopeptídeos/farmacologia , Ratos , Ratos Endogâmicos WKY , Receptor Tipo 1 de Angiotensina/fisiologia , Receptor Tipo 2 de Angiotensina/agonistas , Receptor Tipo 2 de Angiotensina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , ATPase Trocadora de Sódio-Potássio/metabolismo
9.
Eur Biophys J ; 40(2): 165-74, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20976596

RESUMO

A significant amount of Ca²+ is contained in secretory mucin granules. Exchange of Ca²+ for monovalent cations drives the process of mucin decondensation and hydration after fusion of granules with the plasma membrane. Here we report direct observation of calcium secretion with a Ca²+ ion-selective electrode (ISE) in response to apical stimulation with ATP from HT29-Cl.16E cells, a subclone of the human colonic cancer cell line HT29. No increase in Ca²+ level was seen for the sister cell line Cl.19A, which lacks mucin granules, or for Cl.16E cells after inhibition of granule fusion with wortmannin. Further, the measured concentration was used to estimate the time-resolved rate of release of Ca²+ from the cell monolayer, by use of a deconvolution-based method developed previously (Nair and Gratzl in Anal Chem 77:2875-2881, 2005). The results argue that Ca²+ release by Cl.16E cells is associated specifically with mucin secretion, i.e., that the measured Ca²+ increase in the apical solution is derived from granules after fusion and mucin exocytosis. The Ca²+ ISE in conjunction with deconvolution provides a minimally disturbing method for assessment of Ca²+ secretion rates. The release rates provide estimates of exocytosis rates and, when combined with earlier capacitance measurements, estimates of post-stimulation endocytosis rates also.


Assuntos
Cálcio/metabolismo , Células Epiteliais/metabolismo , Mucinas/metabolismo , Técnicas de Patch-Clamp/métodos , Trifosfato de Adenosina/metabolismo , Cátions Bivalentes/metabolismo , Endocitose , Exocitose , Células HT29 , Humanos , Eletrodos Seletivos de Íons
11.
Am J Physiol Renal Physiol ; 297(5): F1342-52, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19726542

RESUMO

Angiotensin II (ANG II) is taken up by proximal tubule (PT) cells via AT1 (AT1a) receptor-mediated endocytosis, but the underlying cellular mechanisms remain poorly understood. The present study tested the hypothesis that the microtubule- rather than the clathrin-dependent endocytic pathway regulates AT1-mediated uptake of ANG II and ANG II-induced sodium and hydrogen exchanger-3 (NHE-3) expression in PT cells. The expression of AT1 receptors, clathrin light (LC) and heavy chain (HC) proteins, and type 1 microtubule-associated proteins (MAPs; MAP-1A and MAP-1B) in PT cells were knocked down by their respective small interfering (si) RNAs before AT1-mediated FITC-ANG II uptake and ANG II-induced NHE-3 expression were studied. AT1 siRNAs inhibited AT1 expression and blocked ANG II-induced NHE-3 expression in PT cells, as expected (P < 0.01). Clathrin LC or HC siRNAs knocked down their respective proteins by approximately 90% with a peak response at 24 h, and blocked the clathrin-dependent uptake of Alexa Fluor 594-transferrin (P < 0.01). However, neither LC nor HC siRNAs inhibited AT1-mediated uptake of FITC-ANG II or affected ANG II-induced NHE-3 expression. MAP-1A or MAP-1B siRNAs markedly knocked down MAP-1A or MAP-1B proteins in a time-dependent manner with peak inhibitions at 48 h (>76.8%, P < 0.01). MAP protein knockdown resulted in approximately 52% decreases in AT1-mediated FITC-ANG II uptake and approximately 66% decreases in ANG II-induced NHE-3 expression (P < 0.01). These effects were associated with threefold decreases in ANG II-induced MAP kinases ERK 1/2 activation (P < 0.01), but not with altered AT1 expression or clathrin-dependent transferrin uptake. Both losartan and AT1a receptor deletion in mouse PT cells completely abolished the effects of MAP-1A knockdown on ANG II-induced NHE-3 expression and activation of MAP kinases ERK1/2. Our findings suggest that the alternative microtubule-dependent endocytic pathway, rather than the canonical clathrin-dependent pathway, plays an important role in AT1 (AT1a)-mediated uptake of extracellular ANG II and ANG II-induced NHE-3 expression in PT cells.


Assuntos
Angiotensina II/metabolismo , Endocitose/fisiologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Receptor Tipo 1 de Angiotensina/fisiologia , Trocadores de Sódio-Hidrogênio/biossíntese , Actinas/biossíntese , Actinas/genética , Angiotensina II/farmacologia , Animais , Western Blotting , Células Cultivadas , Clatrina/biossíntese , Proteínas do Citoesqueleto/biossíntese , Proteínas do Citoesqueleto/genética , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Indóis , Túbulos Renais Proximais/citologia , Camundongos , Microscopia de Fluorescência , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , RNA Interferente Pequeno/farmacologia , Coelhos , Receptor Tipo 1 de Angiotensina/biossíntese , Receptor Tipo 1 de Angiotensina/genética , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genética
12.
Hypertension ; 53(6): 1054-61, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19380616

RESUMO

NADPH oxidase (Nox)-dependent reactive oxygen species production is implicated in the pathogenesis of cardiovascular diseases, including hypertension. We tested the hypothesis that oxidase subunits are differentially regulated in renal proximal tubules from normotensive and spontaneously hypertensive rats. Basal Nox2 and Nox4, but not Rac1, in immortalized renal proximal tubule cells and brush border membranes were greater in hypertensive than in normotensive rats. However, more Rac1 was expressed in lipid rafts in cells from hypertensive rats than in cells from normotensive rats; the converse was observed with Nox4, whereas Nox2 expression was similar. The D(1)-like receptor agonist fenoldopam decreased Nox2 and Rac1 protein in lipid rafts to a greater extent in hypertensive than in normotensive rats. Basal oxidase activity was 3-fold higher in hypertensive than in normotensive rats but was inhibited to a greater extent by fenoldopam in normotensive (58+/-3.3%) than in hypertensive rats (31+/-5.2%; P<0.05; n=6 per group). Fenoldopam decreased the amount of Nox2 that coimmunoprecipitated with p67(phox) in cells from normotensive rats. D(1)-like receptors may decrease oxidase activity by disrupting the distribution and assembly of oxidase subunits in cell membrane microdomains. The cholesterol-depleting reagent methyl-beta-cyclodextrin decreased oxidase activity and cholesterol content to a greater extent in hypertensive than in normotensive rats. The greater basal levels of Nox2 and Nox4 in cell membranes and Nox2 and Rac1 in lipid rafts in hypertensive rats than in normotensive rats may explain the increased basal oxidase activity in hypertensive rats.


Assuntos
Túbulos Renais Proximais/citologia , Microdomínios da Membrana/metabolismo , NADPH Oxidases/metabolismo , Receptores de Dopamina D1/metabolismo , Análise de Variância , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Fenoldopam/farmacologia , Immunoblotting , Luminescência , NADPH Oxidases/genética , Probabilidade , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Espécies Reativas de Oxigênio/metabolismo , Receptores de Dopamina D1/agonistas , Sensibilidade e Especificidade
13.
Am J Hypertens ; 22(8): 877-83, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19390510

RESUMO

BACKGROUND: The dopaminergic and endothelin systems, by regulating sodium transport in the renal proximal tubule (RPT), participate in the control of blood pressure. The D(3) and ETB receptors are expressed in RPTs, and D(3) receptor function in RPTs is impaired in spontaneously hypertensive rats (SHRs). Therefore, we tested the hypothesis that D(3) receptors can regulate ETB receptors, and that D(3) receptor regulation of ETB receptors in RPTs is impaired in SHRs. METHODS: ETB receptor expression in RPT cells was measured by immunoblotting and reverse transcriptase-PCR and ETB receptor function by measuring Na(+)-K(+) ATPase activity. D(3)/ETB receptor interaction was studied by co-immunoprecipitation. RESULTS: In Wistar-Kyoto (WKY) RPT cells, the D(3) receptor agonist, PD128907, increased ETB receptor protein expression, effects that were blocked by removal of calcium in the culture medium. The stimulatory effect of D(3) on ETB receptor mRNA and protein expression was also blocked by nicardipine. In contrast, in SHR RPT cells, PD128907 decreased ETB receptor expression. Basal D(3)/ETB receptor co-immunoprecipitation was three times greater in WKY than in SHRs. The absolute amount of D(3)/ETB receptor co-immunoprecipitation induced by a D(3) receptor agonist was also greater in WKY than in SHRs. Stimulation of ETB receptors decreased Na(+)-K(+) ATPase activity in WKY but not in SHR cells. Pretreatment with PD128907 augmented the inhibitory effect of BQ3020 on Na(+)-K(+) ATPase activity in WKY but not in SHR cells. CONCLUSIONS: D(3) receptors regulate ETB receptors by physical receptor interaction and govern receptor expression and function. D(3) receptor regulation of ETB receptors is aberrant in RPT cells from SHRs.


Assuntos
Túbulos Renais Proximais/fisiologia , Receptor de Endotelina B/fisiologia , Receptores de Dopamina D3/fisiologia , Animais , Benzopiranos/farmacologia , Western Blotting , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Proteínas de Ligação ao GTP/fisiologia , Imunoprecipitação , Indanos/farmacologia , Túbulos Renais Proximais/citologia , Nicardipino/farmacologia , Oxazinas/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptor de Endotelina B/genética , Receptores de Dopamina D3/agonistas , Receptores de Dopamina D3/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sistemas do Segundo Mensageiro/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Especificidade da Espécie
14.
Am J Hypertens ; 22(7): 770-6, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19373217

RESUMO

BACKGROUND: Ion transport in the renal proximal tubule (RPT) is regulated by numerous hormones and humoral factors, including insulin and dopamine. Previous studies show an interaction between insulin and the D(1) receptor. Because both D(1) and D(5) receptors belong to the D(1)-like receptor subfamily, it is possible that an interaction between insulin and the D(5) dopamine receptor exists in RPT cells from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). METHODS: D(5) receptor expression in immortalized RPT cells from WKY and SHRs was quantified by immunoblotting and D(5) receptor function by measuring Na(+)-K(+) ATPase activity. RESULTS: Insulin increased the expression of the D(5) receptor. Stimulation with insulin (10(-7) mol/l) for 24 h increased D(5) receptor expression in RPT cells from WKY rats. This effect of insulin on D(5) receptor expression was aberrant in RPT cells from SHRs. The stimulatory effect of insulin on D(5) receptor expression in RPT cells from WKY rats was inhibited by a protein kinase C (PKC) inhibitor (PKC inhibitor peptide 19-31, 10(-6) mol/l) or a phosphatidylinositol 3 (PI3) kinase inhibitor (wortmannin, 10(-6) mol/l), indicating that both PKC and PI3 kinase were involved in the signaling pathway. Stimulation of the D(5) receptor heterologously expressed in HEK293 cells with fenoldopam (10(-7) mol/l/15 min) inhibited Na(+)-K(+) ATPase activity, whereas pretreatment with insulin (10(-7) mol/l/24 h) increased the D(5) receptor-mediated inhibition. CONCLUSIONS: Insulin and D(5) receptors interact to regulate renal sodium transport; an aberrant interaction between insulin and D(5) receptor may participate in the pathogenesis of hypertension.


Assuntos
Insulina/farmacologia , Túbulos Renais Proximais/metabolismo , Receptores de Dopamina D5/fisiologia , Animais , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptor de Insulina/metabolismo , Receptores de Dopamina D5/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Estimulação Química
15.
Kidney Int ; 74(6): 750-9, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18547994

RESUMO

Dopaminergic and endothelin systems participate in the control blood pressure by regulating sodium transport in the renal proximal tubule. Disruption of either the endothelin B receptor (ETB) or D(3) dopamine receptor gene in mice produces hypertension. To examine whether these two receptors interact we studied the Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats by selectively infusing reagents into the right kidney of anesthetized rats. The D(3) receptor agonist (PD128907) caused natriuresis in WKY rats which was partially blocked by the ETB receptor antagonist. In contrast, PD128907 blunted sodium excretion in the SHRs. We found using laser confocal microscopy that the ETB receptor was mainly located in the cell membrane in control WKY cells. Treatment with the D(3) receptor antagonist caused its internalization into intracellular compartments that contained the D(3) receptors. Combined use of D(3) and ETB antagonists failed to internalize ETB receptors in cells from WKY rats. In contrast in SHR cells, ETB receptors were found mainly in internal compartments under basal condition and thus were likely prevented from interacting with the agonist-stimulated, membrane-bound D(3) receptors. Our studies suggest that D(3) receptors physically interact with proximal tubule ETB receptors and that the blunted natriuretic effect of dopamine in SHRs may be explained, in part, by abnormal D(3)/ETB receptor interactions.


Assuntos
Rim/metabolismo , Natriurese , Receptor de Endotelina B/metabolismo , Receptores de Dopamina D3/agonistas , Receptores de Dopamina D3/metabolismo , Animais , Membrana Celular/química , Endocitose , Antagonistas do Receptor de Endotelina B , Túbulos Renais Proximais/química , Natriurese/efeitos dos fármacos , Ligação Proteica , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptor de Endotelina B/análise , Receptores de Dopamina D3/antagonistas & inibidores
16.
Hypertension ; 51(5): 1332-8, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18391104

RESUMO

The present study tested the hypothesis that angiotensin II (Ang II)-induced oxidative stress and Ang II-stimulated Cl(-)/HCO(3)(-) exchanger are increased and related to the differential membrane Ang II type 1 (AT(1)) receptor and reduced nicotinamide-adenine dinucleotide phosphate oxidase expression in immortalized renal proximal tubular epithelial (PTE) cells from the spontaneously hypertensive rat (SHR) relative to its normotensive control (Wistar Kyoto rat [WKY]). The exposure of cells to Ang II increased Cl(-)/HCO(3)(-) exchanger activity with EC(50)s of 0.10 and 12.2 nmol/L in SHR and WKY PTE cells, respectively. SHR PTE cells were found to overexpress nicotinamide-adenine dinucleotide phosphate oxidase 2 and 4 and were endowed with an enhanced ability to generate H(2)O(2). The reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor apocynin reduced the production of H(2)O(2) in SHR PTE cells and abolished their hypersensitivity to Ang II. The expression of the glycosylated form of the AT(1) receptor in both lipid and nonlipid rafts were higher in SHR cells than in WKY PTE cells. Pretreatment with apocynin reduced the abundance of AT(1) receptors in both microdomains, mainly the glycosylated form of the AT(1) receptor in lipid rafts, in SHR cells but not in WKY PTE cells. In conclusion, differences between WKY and SHR PTE cells in their sensitivity to Ang II correlate with the higher H(2)O(2) generation that provokes an enhanced expression of glycosylated and nonglycosylated AT(1) receptor forms in lipid rafts.


Assuntos
Angiotensina II/farmacologia , Antiportadores de Cloreto-Bicarbonato/metabolismo , Peróxido de Hidrogênio/metabolismo , Túbulos Renais Proximais/metabolismo , Microdomínios da Membrana/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Acetofenonas/farmacologia , Animais , Antioxidantes/farmacologia , Membrana Celular/metabolismo , Membrana Celular/patologia , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Hipertensão/metabolismo , Hipertensão/patologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Microdomínios da Membrana/patologia , NADPH Oxidases/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Transdução de Sinais/fisiologia
17.
Biochem Pharmacol ; 75(11): 2224-33, 2008 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-18407247

RESUMO

The present study evaluated the activity of Cl(-)/HCO(3)(-) exchanger and the abundance of Slc26a6 in immortalized renal proximal tubular epithelial (PTE) cells from the Wistar-Kyoto rat (WKY) and spontaneously hypertensive rat (SHR) and identified the signaling pathways that regulate the activity of the transporter. The affinity for HCO(3)(-) was identical in WKY and SHR PTE cells, but V(max) values (in pH units/min) in SHR PTE cells (0.4016) were significantly higher than in WKY PTE cells (0.2304). The expression of Slc26a6 in SHR PTE cells was sevenfold that in WKY PTE cells. Dibutyryl-cAMP (db-cAMP) or forskolin, which increased endogenous cAMP, phorbol-12,13-dibutyrate (PDBu) and anisomycin, significantly (P<0.05) increased the Cl(-)/HCO(3)(-) exchanger activity in WKY and SHR PTE cells to a similar extent. The stimulatory effects of db-cAMP and forskolin were prevented by the PKA inhibitor H89, but not by chelerythrine. The stimulatory effects of PDBu were prevented by both chelerythrine and SB 203580, but not by H89 or the MEK inhibitor PD 98059. The stimulatory effect of anisomycin was prevented by SB 203580, but not by chelerythrine. Increases in phospho-p38 MAPK by anisomycin were identical in WKY and SHR PTE cells, this being sensitive to SB 203580 but not to chelerythrine. It is concluded that SHR PTE cells, which overexpress the Slc26a6 protein, are endowed with an enhanced activity of the Cl(-)/HCO(3)(-) exchanger. The Cl(-)/HCO(3)(-) exchanger is an effector protein for PKA, PKC and p38 MAPK in both WKY and SHR PTE cells.


Assuntos
Antiportadores de Cloreto-Bicarbonato/metabolismo , Células Epiteliais/metabolismo , Túbulos Renais Proximais/citologia , Animais , Linhagem Celular , Antiportadores de Cloreto-Bicarbonato/genética , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY
18.
Mol Cell Biochem ; 310(1-2): 191-201, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18095144

RESUMO

This study evaluated the effects of aldosterone upon Na+/H+ exchange (NHE) activity in immortalized proximal tubular epithelial (PTE) cells from the spontaneously hypertensive rat (SHR) and the normotensive controls (Wistar Kyoto rat; WKY). Increases in NHE activity after exposure to aldosterone occurred in time- and concentration-dependent manner in SHR PTE cells, but not in WKY PTE cells. The aldosterone-induced increases in NHE activity were prevented by spironolactone, but not by the glucocorticoid receptor antagonist Ru 38486. The presence of the mineralocorticoid receptor transcript was confirmed by PCR and NHE1, NHE2, and NHE3 proteins were detected by immunoblot analysis. Cariporide and EIPA, but not S3226, inhibited the aldosterone-induced increase in NHE activity, indicating that NHE1 is the most likely involved NHE isoform. Pretreatment of SHR PTE cells with actinomycin D attenuated the aldosterone-induced increases in NHE activity. The SHR PTE cells had an increased rate of H2O2 production when compared with WKY PTE cells. Treatment of cells with apocynin, a NADPH oxidase inhibitor, markedly reduced the rate of H2O2 production. The aldosterone-induced increase in NHE activity SHR PTE cells was completely prevented by apocynin. In conclusion, the aldosterone-induced stimulation of NHE1 activity is a genomic event unique in SHR PTE cells, which involves the activation of the mineralocorticoid receptor, but ultimately requires the availability of H2O2 in excess.


Assuntos
Aldosterona/farmacologia , Genoma/genética , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Trocadores de Sódio-Hidrogênio/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacologia , Animais , Linhagem Celular , Citocalasina B/farmacologia , Dactinomicina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Guanidinas/farmacologia , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Immunoblotting , Túbulos Renais Proximais/efeitos dos fármacos , Metacrilatos/farmacologia , Mifepristona/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos SHR , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Trocador 1 de Sódio-Hidrogênio , Espironolactona/farmacologia , Sulfonas/farmacologia
19.
Eur Biophys J ; 37(4): 411-9, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17968537

RESUMO

Short-circuit current (Isc) measurement is used to quantify transepithelial ion flux. This technique provides a direct measure of net charge transport across a cell monolayer. Isc however, lacks chemical selectivity. Chemically resolved ion fluxes may be much greater than Isc, and differ in different biological processes. This work describes a novel experimental approach and deconvolution method to obtain temporally resolved ion fluxes at epithelial cell monolayers. HT29-Cl.16E cells, a sub clone of the human colonic cancer cell line HT29 was used as a model cell line to validate this approach in the context of epithelial transport studies. This cell line is known to secrete chloride in response to purinergic stimulation. Changes in chloride concentration after stimulation with 1 mM ATP plus 50 nM phorbol-myristate acetate (PMA) are recorded with a chloride ion-selective electrode (ISE) at a short distance (approximately 50 microm) from the monolayer. The recorded concentrations are transformed to corresponding chloride flux across the monolayer using a deconvolution algorithm for extracellular mass transport based on minimization of the shape error function (Nair and Gratzl in Anal Chem 77:2875-2888, 2005). Simultaneous voltage clamp yields the associated net electrical charge flux (Isc). The dynamics of Cl(-) flux did correlate with that of the electrical flux, but was found to be greater in amplitude. This suggests that Cl(-) may not be the only ion secreted. The method of simultaneously assessing ionic and electrical fluxes with a temporal resolution of seconds provides unique information about the dynamics of solute fluxes across the apical membrane.


Assuntos
Cloretos/química , Células Epiteliais/metabolismo , Mucinas/química , Transporte Biológico , Transporte Biológico Ativo , Linhagem Celular Tumoral , Canais de Cloreto , Eletrodos , Eletrofisiologia/métodos , Epitélio/patologia , Desenho de Equipamento , Humanos , Transporte de Íons , Íons , Fatores de Tempo
20.
Front Biosci ; 13: 1665-80, 2008 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17981658

RESUMO

The ciliary/flagellar system is perhaps unique in biology in that not only are biochemical manipulations used to elucidate the function, but physical manipulations as well. Thus, there is a considerable need to have an integrated physical-biochemical model of a cilium and its function. The emphasis of this paper will be to firstly, provide a mechanistic picture of the cilium and its environment because the biological community is perhaps less aware of this type of model development, and second, to point the way towards future experiments that will elucidate the role of the cilium in organ and organism level signaling and regulation.


Assuntos
Cílios/fisiologia , Animais , Bactérias/metabolismo , Cílios/metabolismo , Humanos , Mecanotransdução Celular , Modelos Biológicos , Modelos Estatísticos , Modelos Teóricos , Estresse Mecânico , Biologia de Sistemas
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