RESUMO
The kidney is an important target of the renin-ANG-aldosterone system (RAAS). To date, several studies have demonstrated the existence of a local RAAS in various tissues, including the renal tissue. The mineralocorticoid aldosterone is known to play a critical role in the classical RAAS; however, its effect on mesangial cells (MCs) remains to be elucidated. Based on this, our aim was to investigate whether aldosterone stimulation can modulate the intracellular RAAS of immortalized human MCs by evaluating ANG-converting enzyme (ACE)/ANG II/ANG II receptor type 1 (AT1) and ANG-converting enzyme 2 (ACE2)/ANG (1-7)/MAS receptor axes. To realise this, protein expression, enzyme activity, and immunofluorescence were performed under aldosterone stimulation and in the presence of the mineralocorticoid receptor (MR) antagonist spironolactone (SPI). We observed that high doses of aldosterone increase ACE activity. The effect of aldosterone on the catalytic activity of ACE was completely abolished with the pretreatment of SPI suggesting that the aldosterone-induced cell injuries through ANG II release were attenuated. Aldosterone treatment also decreased the expression of MAS receptor, but did not alter the expression or the catalytic activity of ACE 2 and ANG (1-7) levels. Spironolactone modulated the localization of ANG II and AT1 receptor and decreased ANG (1-7) and MAS receptor levels. Our data suggest that both aldosterone and the MR receptor antagonist can modulate both of these axes and that spironolactone can protect MCs from the damage induced by aldosterone.
Assuntos
Aldosterona/farmacologia , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/metabolismo , Espironolactona/farmacologia , Angiotensina I/genética , Angiotensina I/metabolismo , Enzima de Conversão de Angiotensina 2 , Células Cultivadas , Glicosilação/efeitos dos fármacos , Humanos , Células Mesangiais/citologia , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Biological effects of angiotensin II (AngII) such as regulation of AngII target genes may be triggered by interaction of AngII with intracellular AngII receptor types 1 and 2 (AT1 and AT2), defined as intracrine response. The aim of this study was to examine the presence of AT1 and AT2 receptors in nuclear membrane of human mesangial cells (HMCs) and evaluate the possible biological effects mediated by intracellular AT1 through an intracrine mechanism. Subcellular distribution of AT1 and AT2 was evaluated by immunofluorescence and by western blot in isolated nuclear extract. Endogenous intracellular synthesis of AngII was stimulated by high glucose (HG). Effects of HG were analyzed in the presence of candesartan, which prevents AngII internalization. Both receptors were found in nuclear membrane. Fluorescein isothiocyanate (FITC)-labeled AngII added to isolated nuclei produced a fluorescence that was reduced in the presence of losartan or PD-123319 and quenched in the presence of both inhibitors simultaneously. HG induced overexpression of fibronectin and increased cell proliferation in the presence of candesartan, indicating an intracrine action of AngII induced by HG. Results showed the presence of nuclear receptors in HMCs that can be activated by AngII through an intracrine response independent of cytoplasmic membrane AngII receptors.
Assuntos
Angiotensina II/metabolismo , Células Mesangiais/metabolismo , Membrana Nuclear/metabolismo , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/metabolismo , Linhagem Celular Transformada , Humanos , Células Mesangiais/citologiaRESUMO
Two new complexes [Cu(Cand)(H2O)4] [1] and [Cu2(TCand)4(H2O)2]·4H2O [2] (Cand = candesartan; TCand = trityl candesartan) have been synthesized and thoroughly characterized. The FTIR, Raman, EPR and diffuse reflectance spectra of the solid compounds show a dimeric complex for [2] with carboxylate bridging of the type found in copper(II) acetate. Both elemental analysis and thermal measurements allow the determination of the total stoichiometries of both complexes. The stability measurements show that the compounds are stable in ethanolic solutions at least for 1h, while the preservation of the overall stochiometry for both species in solution has been determined by spectrophotometric titrations. By metal complexation the absence of antioxidant behavior of both sartans has been improved. Complexes [1] and [2] are strong superoxidedismutase mimetic compounds and complex [2] also behaves as a peroxyl radical scavenger. Furthermore, this higher antioxidant activity works in parallel with the improvement of the expansive activity over the angiotensin II-induced contracted human mesangial cells. These new complexes exhibit even higher efficiency as drugs in comparison with the free non-complexed medication with increased antioxidant ability expressing higher capacity to block the angiotensin II contractile effect. This study provides a new insight into the development of copper(II) complexes as potential drugs.
Assuntos
Anti-Hipertensivos/farmacologia , Benzimidazóis/química , Benzimidazóis/farmacologia , Cobre/química , Tetrazóis/química , Tetrazóis/farmacologia , Angiotensina II/farmacologia , Anti-Hipertensivos/química , Antioxidantes/metabolismo , Compostos de Bifenilo , Humanos , Células Mesangiais/citologia , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/metabolismo , Estrutura Molecular , Espectroscopia de Infravermelho com Transformada de Fourier , Superóxido Dismutase/metabolismoRESUMO
The prorenin receptor [(P)RR] is upregulated in the diabetic kidney and has been implicated in the high glucose (HG)-induced overproduction of profibrotic molecules by mesangial cells (MCs), which is mediated by ERK1/2 phosphorylation. The regulation of (P)RR gene transcription and the mechanisms by which HG increases (P)RR gene expression are not fully understood. Because intracellular levels of angiotensin II (AngII) are increased in MCs stimulated with HG, we used this in vitro system to evaluate the possible role of AngII in (P)RR gene expression and function by comparing the effects of AT1 receptor blockers (losartan or candesartan) and (P)RR mRNA silencing (siRNA) in human MCs (HMCs) stimulated with HG. HG induced an increase in (P)RR and fibronectin expression and in ERK1/2 phosphorylation. These effects were completely reversed by (P)RR siRNA and losartan but not by candesartan (an angiotensin receptor blocker that, in contrast to losartan, blocks AT1 receptor internalization). These results suggest that (P)RR gene activity may be controlled by intracellular AngII and that HG-induced ERK1/2 phosphorylation and fibronectin overproduction are primarily induced by (P)RR activation. This relationship between AngII and (P)RR may constitute an additional pathway of MC dysfunction in response to HG stimulation.
Assuntos
Angiotensina II/metabolismo , Receptores de Superfície Celular/metabolismo , Sistema Renina-Angiotensina , ATPases Vacuolares Próton-Translocadoras/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fibronectinas/genética , Fibronectinas/metabolismo , Inativação Gênica/efeitos dos fármacos , Glucose/farmacologia , Humanos , Losartan/farmacologia , Células Mesangiais/citologia , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores de Superfície Celular/genética , Sistema Renina-Angiotensina/efeitos dos fármacos , Fatores de Tempo , Azul Tripano/metabolismo , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
The proliferation of mesangial cells (MC) in the presence of glutamine (0-20 mM) was determined in both low (5 mM) and high (25 mM) glucose-containing medium. Glutamine in a high glucose (HG) environment increased cell proliferation in a dose-dependent manner. Inhibition of glutamine:fructose 6-phosphate amidotransferase (GFAT) and of phosphodiesterase significantly reduced glutamine-induced proliferation. Supraphysiologic levels of glutamine increase MC proliferation in a HG milieu via GFAT and cAMP-dependent pathways, suggesting that glutamine could pose a risk for diabetic nephropathy.
Assuntos
Glucose/farmacologia , Glutamina/farmacologia , Células Mesangiais/citologia , Células Mesangiais/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/antagonistas & inibidores , Humanos , Diester Fosfórico Hidrolases/efeitos dos fármacosRESUMO
BACKGROUND/AIMS: Previous reports have shown the presence of streptococcal erythrogenic toxin type B (ETB), IL-8, transforming growth factor-beta (TGF-beta) and glomerular proliferation in renal biopsies from patients with acute poststreptococcal glomerulonephritis (APSGN). In addition, increased levels of plasma IL-6 and tumor necrosis factor-alpha (TNFalpha) and urinary IL-6 have also been reported in this disease. To determine the effect of ETB in mesangial cell cytokine production and proliferation, the concentration of several cytokines (IL-6, IL-1beta, TNFalpha, IL-10, IL-4, RANTES), soluble TNF receptor I (STNFR-I), soluble TNF receptor II (STNFR-II) and proliferation were measured in rat mesangial cells cultures after treatment with ETB or its precursor (ETBP). METHODS: To analyze the levels of cytokines and production of soluble receptors as well as proliferation, rat mesangial cells were cultured with ETB or ETBP (50 microg/ml). After 24, 48 and 96 h of incubation, culture supernatants were assessed for cytokines and receptors by ELISA and for proliferation by incorporation of radioactive thymidine. RESULTS: A significant increase in IL-6 levels was found in mesangial cell cultures treated with either ETBP or ETB when compared with controls. Streptococcal proteins treated mesangial cells also showed elevated levels of proliferation at 96 h. Increased production of IL-6 was not correlated with proliferation. A polyclonal anti-ETB antibody abolished the IL-6 stimulatory effect of ETB on mesangial cells. ETB/ETBP failed to increase the levels of other cytokines and cytokine soluble receptors. CONCLUSION: Streptococcal ETB/ETBP is capable of inducing increased production of IL-6 and proliferation on mesangial cells. These findings could be relevant in a possible early interaction of streptococcal proteins with mesangial cells and during the course of APSGN.
Assuntos
Proteínas de Bactérias/farmacologia , Exotoxinas/farmacologia , Interleucina-6/análise , Células Mesangiais/química , Células Mesangiais/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/análise , Células Mesangiais/citologia , Ratos , Ratos Sprague-Dawley , Receptores do Fator de Necrose Tumoral/análiseRESUMO
The importance of glutamine for the synthesis of proteins of the extracellular matrix was investigated in cultured mesangial cells. Glutamine at 2 mM elicited an increase in smooth muscle cell alpha-actin, alpha(1)-type IV collagen and fibronectin transcripts (19.0-, 16.7-, and 4.3-fold, respectively) accompanied by an increase in alpha-actin stress fibres compared to cells grown in absence of glutamine. The specificity for the glutamine requirement is suggested by the fact that mRNA levels of tenascin were not altered by addition of glutamine. This suggests that glutamine is required for the expression of important proteins of the extracellular matrix in cultured mesangial cells.