RENATO DE OLIVEIRA CRAJOINAS

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Instituto do Coração, Hospital das Clínicas, Faculdade de Medicina
LIM/13 - Laboratório de Genética e Cardiologia Molecular, Hospital das Clínicas, Faculdade de Medicina

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  • conferenceObject
    Angiotensin II decreases the levels of PKA-mediated NHE3 phosphorylation in renal proximal tubule
    (2012) ROSSETTO, Silmara Larissa; QUEIROZ-LEITE, Gabriella Duarte; CRAJOINAS, Renato Oliveira; OMAE, Samantha V.; MALNIC, Gerhard; GIRARDI, Adriana C.
    NHE3 transport function is inhibited by PKA. Binding of angiotensin II (ANG II) to AT1 receptor has been reported to activate Gi-protein signaling pathway, resulting in the inhibition of adenylyl cyclase, and consequently, in decreased PKA activation. We therefore tested the hypothesis that decrement of PKA-mediated NHE3 phosphorylation is one of the mechanisms by which ANG II acutely stimulates NHE3 activity in renal proximal tubule. To this end, OKP cells were treated with 100 μM Dopamine (DOP) for 30 min in the presence or absence of 10–10 M ANG II for 5, 15, and 30 min; or only with ANG II for 30 min. Extrusion of H+ from OKP cells was measured. DOP significantly inhibited NHE3 activity (0.2013 ± 0.009 vs. 0.3036 ± 0.0019 pH units/min in control). Simultaneous incubation of DOP with ANG II for 15 (0.2347 ± 0.044 pH units/min) and 30 min (0.3295 ± 0.032 pH units/min) completely blocked this inhibitory effect. As expected, ANG II stimulated NHE3 activity. DOP increased the levels of NHE3 phosphorylated at the PKA consensus phosphorylation sites [serines 552 and 605] by 64 ± 8% and 288 ± 37% respectively vs. control. ANG II treatment for 15 and 30 min prevented this increase. PKA activity was also measured in these groups of cells. We found that PKA activity was significantly increased in cells treated with DOP for 30 min and in cells treated with DOP plus ANG II for 5 min. Longer incubation times with ANG II (15 and 30 min) in the presence of DOP restored PKA activity near to control levels. Losartan blocked all the effects of ANG II here reported. Collectively, these data suggest that inhibition of PKA activity leading to lower levels of endogenous phosphorylation of NHE3 is one of the mechanisms by which ANG II stimulates NHE3 activity in the proximal tubule.
  • article 192 Citação(ões) na Scopus
    Mechanisms mediating the diuretic and natriuretic actions of the incretin hormone glucagon-like peptide-1
    (2011) CRAJOINAS, Renato O.; ORICCHIO, Felipe T.; PESSOA, Thaissa D.; PACHECO, Bruna P. M.; LESSA, Lucilia M. A.; MALNIC, Gerhard; GIRARDI, Adriana C. C.
    Crajoinas RO, Oricchio FT, Pessoa TD, Pacheco BP, Lessa LM, Malnic G, Girardi AC. Mechanisms mediating the diuretic and natriuretic actions of the incretin hormone glucagon-like peptide-1. Am J Physiol Renal Physiol 301: F355-F363, 2011. First published May 18, 2011; doi: 10.1152/ajprenal.00729.2010.-Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone considered a promising therapeutic agent for type 2 diabetes because it stimulates beta cell proliferation and insulin secretion in a glucose-dependent manner. Cumulative evidence supports a role for GLP-1 in modulating renal function; however, the mechanisms by which GLP-1 induces diuresis and natriuresis have not been completely established. This study aimed to define the cellular and molecular mechanisms mediating the renal effects of GLP-1. GLP-1 (1 mu g.kg(-1).min(-1)) was intravenously administered in rats for the period of 60 min. GLP-1-infused rats displayed increased urine flow, fractional excretion of sodium, potassium, and bicarbonate compared with those rats that received vehicle (1% BSA/saline). GLP-1-induced diuresis and natriuresis were also accompanied by increases in renal plasma flow and glomerular filtration rate. Real-time RT-PCR in microdissected rat nephron segments revealed that GLP-1 receptor-mRNA expression was restricted to glomerulus and proximal convoluted tubule. In rat renal proximal tubule, GLP-1 significantly reduced Na+/H+ exchanger isoform 3 (NHE3)-mediated bicarbonate reabsorption via a protein kinase A (PKA)-dependent mechanism. Reduced proximal tubular bicarbonate flux rate was associated with a significant increase of NHE3 phosphorylation at the PKA consensus sites in microvillus membrane vesicles. Taken together, these data suggest that GLP-1 has diuretic and natriuretic effects that are mediated by changes in renal hemodynamics and by downregulation of NHE3 activity in the renal proximal tubule. Moreover, our findings support the view that GLP-1-based agents may have a potential therapeutic use not only as antidiabetic drugs but also in hypertension and other disorders of sodium retention.
  • article 27 Citação(ões) na Scopus
    Amelioration of Cardiac Function and Activation of Anti-Inflammatory Vasoactive Peptides Expression in the Rat Myocardium by Low Level Laser Therapy
    (2014) MANCHINI, Martha Trindade; SERRA, Andrey Jorge; FELICIANO, Regiane dos Santos; SANTANA, Eduardo Tadeu; ANTONIO, Ednei Luis; CARVALHO, Paulo de Tarso Camillo de; MONTEMOR, Jairo; CRAJOINAS, Renato Oliveira; GIRARDI, Adriana Castello Costa; TUCCI, Paulo Jose Ferreira; SILVA JR., Jose Antonio
    Low-level laser therapy (LLLT) has been used as an anti-inflammatory treatment in several disease conditions, even when inflammation is a secondary consequence, such as in myocardial infarction (MI). However, the mechanism by which LLLT is able to protect the remaining myocardium remains unclear. The present study tested the hypothesis that LLLT reduces inflammation after acute MI in female rats and ameliorates cardiac function. The potential participation of the Renin-Angiotensin System (RAS) and Kallikrein-Kinin System (KKS) vasoactive peptides was also evaluated. LLLT treatment effectively reduced MI size, attenuated the systolic dysfunction after MI, and decreased the myocardial mRNA expression of interleukin-1 beta and interleukin-6 in comparison to the non-irradiated rat tissue. In addition, LLLT treatment increased protein and mRNA levels of the Mas receptor, the mRNA expression of kinin B2 receptors and the circulating levels of plasma kallikrein compared to non-treated post-MI rats. On the other hand, the kinin B1 receptor mRNA expression decreased after LLLT. No significant changes were found in the expression of vascular endothelial growth factor (VEGF) in the myocardial remote area between laser-irradiated and non-irradiated post-MI rats. Capillaries density also remained similar between these two experimental groups. The mRNA expression of the inducible nitric oxide synthase (iNOS) was increased three days after MI, however, this effect was blunted by LLLT. Moreover, endothelial NOS mRNA content increased after LLLT. Plasma nitric oxide metabolites (NOx) concentration was increased three days after MI in non-treated rats and increased even further by LLLT treatment. Our data suggest that LLLT diminishes the acute inflammation in the myocardium, reduces infarct size and attenuates left ventricle dysfunction post-MI and increases vasoactive peptides expression and nitric oxide (NO) generation.
  • article 4 Citação(ões) na Scopus
    Swimming Training Improves Myocardial Mechanics, Prevents Fibrosis, and Alters Expression of Ca2+ Handling Proteins in Older Rats
    (2018) YOSHIZAKI, Amanda; ANTONIO, Ednei Luiz; SILVA JUNIOR, Jose Antonio; CRAJOINAS, Renato Oliveira; SILVA, Flavio Andre; GIRARDI, Adriana Castello Costa; BOCALINI, Danilo Sales; PORTES, Leslie Andrews; SANTOS, Luis Felipe Neves dos; CARLOS, Fernando Pereira; CARVALHO, Paulo de Tarso Camillo de; TUCCI, Paulo Jose Ferreira; SERRA, Andrey Jorge
    Exercise training effects on the contractility of aged myocardium have been investigated for more than 20 years, but the data are still unclear. This study evaluated the hypothesis that a swimming training (ST) may improve myocardial inotropism in older rats. Male Wistar rats aged 4 (young)-and 21 (old)-months-old were divided into young untrained (YNT), old untrained (ONT), and old trained (OTR; 6 weeks of ST) groups. Echocardiography and hemodynamic were employed to assess left ventricular morphology and function. Myocardial mechanics was evaluated on papillary muscles. Histological and immunoblotting were carried out to evaluate fibrosis and proteins that modulate the myocardial function and calcium handling. We found that older rats did not show cardiac dysfunction, but ONT group showed lower physical performance during a swimming test (YNT: 5 +/- 2; ONT: -16 +/- 0.4; OTR: 51 +/- 3; Delta%, sec). Moreover, ONT group showed worse myocardial inotropism, in which it was reversed by ST (Peak developed tension: YNT: 6.2 +/- 0.7; ONT: 3.9 +/- 0.3; OTR: 6.9 +/- 0.9; g/mm(2)). The ST was associated with preserved collagen content (YNT: 0.38 +/- 0.05; ONT: 0.78 +/- 0.12; OTR: 0.34 +/- 0.09; %). Exercise partially mitigated the effects of aging on intracellular Ca2+-regulating protein (eg, L-Ca2+ channel and phospholamban) and beta-isoform of myosin. Thus, we propose that these molecular alterations together with inhibition of collagen increase contribute to improved myocardial performance in older rats.
  • conferenceObject
    Empagliflozin Inhibits NHE3 Activity in the Proximal Tubule of Normotensive and Hypertensive Rats
    (2018) BORGES, Flavio Araujo; SILVA, Corina Albuquerque; CRAJOINAS, Renato Oliveira; CASTELO-BRANCO, Regiane Cardoso; LUCHI, Weverton Machado; GIRARDI, Adriana Castello Costa
  • article 27 Citação(ões) na Scopus
    Fructose Acutely Stimulates NHE3 Activity in Kidney Proximal Tubule
    (2012) QUEIROZ-LEITE, Gabriella D.; CRAJOINAS, Renato O.; NERI, Elida A.; BEZERRA, Camila N. A.; GIRARDI, Adriana C. C.; REBOUCAS, Nancy Amaral; MALNIC, Gerhard
    Background/Aims: Fructose causes a sodium-sensitive hypertension and acutely reduces the urinary Na+ excretion, suggesting that it may regulate the activity of renal tubular sodium transporters. NHE3 is highly expressed in proximal tubule (PT), along with proteins that mediate fructose transport and metabolism. The present work was outlined to investigate whether fructose modulates proximal NHE3 activity and to elucidate the molecular mechanisms underlying this modulation. Methods/Results: Using in vivo stationary microperfusion, we observed that fructose stimulates NHE3 mediated JHCO(3)(-) reabsorption. The MAPK pathway is not involved in this activation, as demonstrated by using of MEK/MAPK inhibitors, whereas experiments using a PKA inhibitor suggest that PKA inhibition plays a role in this response. These results were confirmed in vitro by measuring the cell pH recovery rate after NH4Cl pulse in LLC-PK1, a pig PT cell line, which showed reduced cAMP levels and NHE3 phosphorylation at serine-552 (PKA consensus site) after fructose treatment. Conclusions: NHE3 activity is stimulated by fructose, which increases proximal tubule Na+ reabsorption. The molecular mechanisms involved in this process are mediated, at least in part, by downregulation of the PKA signaling pathway. Future studies are needed to address whether fructose-stimulated NHE3 activity may contribute to renal injury and hypertension.
  • article 25 Citação(ões) na Scopus
    Mechanisms underlying the inhibitory effects of uroguanylin on NHE3 transport activity in renal proximal tubule
    (2012) LESSA, Lucilia M. A.; CARRARO-LACROIX, Luciene R.; CRAJOINAS, Renato O.; BEZERRA, Camila N.; DARIOLLI, Rafael; GIRARDI, Adriana C. C.; FONTELES, Manasses C.; MALNIC, Gerhard
    Lessa LM, Carraro-Lacroix LR, Crajoinas RO, Bezerra CN, Dariolli R, Girardi AC, Fonteles MC, Malnic G. Mechanisms underlying the inhibitory effects of uroguanylin on NHE3 transport activity in renal proximal tubule. Am J Physiol Renal Physiol 303: F1399-F1408, 2012. First published September 5, 2012; doi: 10.1152/ajprenal.00385.2011.-We previously demonstrated that uroguanylin (UGN) significantly inhibits Na+/H+ exchanger (NHE)3-mediated bicarbonate reabsorption. In the present study, we aimed to elucidate the molecular mechanisms underlying the action of UGN on NHE3 in rat renal proximal tubules and in a proximal tubule cell line (LLC-PK1). The in vivo studies were performed by the stationary microperfusion technique, in which we measured H+ secretion in rat renal proximal segments, through a H+-sensitive microelectrode. UGN (1 mu M) significantly inhibited the net of proximal bicarbonate reabsorption. The inhibitory effect of UGN was completely abolished by either the protein kinase G (PKG) inhibitor KT5823 or by the protein kinase A (PKA) inhibitor H-89. The effects of UGN in vitro were found to be similar to those obtained by microperfusion. Indeed, we observed that incubation of LLC-PK1 cells with UGN induced an increase in the intracellular levels of cAMP and cGMP, as well as activation of both PKA and PKG. Furthermore, we found that UGN can increase the levels of NHE3 phosphorylation at the PKA consensus sites 552 and 605 in LLC-PK1 cells. Finally, treatment of LLC-PK1 cells with UGN reduced the amount of NHE3 at the cell surface. Overall, our data suggest that the inhibitory effect of UGN on NHE3 transport activity in proximal tubule is mediated by activation of both cGMP/PKG and cAMP/PKA signaling pathways which in turn leads to NHE3 phosphorylation and reduced NHE3 surface expression. Moreover, this study sheds light on mechanisms by which guanylin peptides
  • article 58 Citação(ões) na Scopus
    Dipeptidyl peptidase IV inhibition upregulates GLUT4 translocation and expression in heart and skeletal muscle of spontaneously hypertensive rats
    (2013) GIANNOCCO, Gisele; OLIVEIRA, Kelen C.; CRAJOINAS, Renato O.; VENTURINI, Gabriela; SALLES, Thiago A.; FONSECA-ALANIZ, Miriam H.; MACIEL, Rui M. B.; GIRARDI, Adriana C. C.
    The purpose of the current study was to test the hypothesis that the dipeptidyl peptidase IV (DPPIV) inhibitor sitagliptin, which exerts anti-hyperglycemic and anti-hypertensive effects, upregulates GLUT4 translocation, protein levels, and/or mRNA expression in heart and skeletal muscle of spontaneously hypertensive rats (SHRs). Ten days of treatment with sitagliptin (40 mg/kg twice daily) decreased plasma DPPIV activity in both young (Y, 5-week-old) and adult (A, 20-week-old) SHRs to similar extents ( similar to 85%). However, DPPIV inhibition only lowered blood pressure in Y-SHRs (119 +/- 3 vs. 136 +/- 4 mmHg). GLUT4 translocation, total protein levels and mRNA expression were decreased in the heart, soleus and gastrocnemius muscle of SHRs compared to age-matched Wistar Kyoto (WKY) normotensive rats. These differences were much more pronounced between A-SHRs and A-WKY rats than between Y-SHRs and Y-WKY rats. In Y-SHRs, sitagliptin normalized GLUT4 expression in the heart, soleus and gastrocnemius. In A-SHRs, sitagliptin increased GLUT4 expression to levels that were even higher than those of A-WKY rats. Sitagliptin enhanced the circulating levels of the DPPIV substrate glucagon-like peptide-1 (GLP-1) in SHRs. In addition, stimulation of the GLP-1 receptor in cardiomyocytes isolated from SHRs increased the protein level of GLUT4 by 154 +/- 13%. Collectively, these results indicate that DPPIV inhibition upregulates GLUT4 in heart and skeletal muscle of SHRs. The underlying mechanism of sitagliptin-induced upregulation of GLUT4 in SHRs may be, at least partially, attributed to GLP-1.
  • article 9 Citação(ões) na Scopus
    Changes in the activity and expression of protein phosphatase-1 accompany the differential regulation of NHE3 before and after the onset of hypertension in spontaneously hypertensive rats
    (2014) CRAJOINAS, R. O.; PESSOA, T. D.; RODRIGUES, M. V.; MALNIC, G.; GIRARDI, A. C. C.
    AimThe Na+/H+ exchanger NHE3 activity decreases in the proximal tubule of spontaneously hypertensive rats (SHRs) as blood pressure increases, and this reduction is correlated with higher NHE3 phosphorylation levels at the PKA consensus site serine 552. This study tested the hypothesis that this lowered NHE3 activity is associated with an increase in PKA activity and expression, and/or a decrease in protein phosphatase-1 (PP1) activity and expression. MethodsProximal tubule NHE3 activity was measured as the rate of bicarbonate reabsorption by stationary microperfusion. NHE3 phosphorylation and protein expression were determined by immunoblotting. PKA and PP1 activities were determined using specific substrates under optimal enzymatic conditions. ResultsThe PKA activator, 6-MB-cAMP, increased the phosphorylation levels of NHE3 at serine 552 in the renal cortex; this increase happens to a much greater extent in young pre-hypertensive SHRs (Y-SHRs) compared to adult SHRs with established hypertension (A-SHRs). Likewise, the inhibitory effect of 6-MB-cAMP on NHE3 transport activity was much more pronounced in the proximal tubules of Y-SHRs than in those of A-SHRs. Renal cortical activity of PKA was not significantly different between Y-SHRs and A-SHRs. On the other hand, Y-SHRs exhibited higher protein phosphatase 1 (PP1) activity, and their expression of the PP1 catalytic subunit PP1 in the renal cortex was also higher than in A-SHRs. ConclusionCollectively, these results support the idea that the lower NHE3 transport activity and higher phosphorylation occurring after the development of hypertension in SHRs are due, at least in part, to reduced PP1-mediated dephosphorylation of NHE3 at serine 552.
  • article 1 Citação(ões) na Scopus
    High blood pressure induced by vitamin D deficiency is associated with renal overexpression and hyperphosphorylation of Na+-K+-2Cl-cotransporter type 2
    (2021) LUCHI, Weverton M.; CRAJOINAS, Renato O.; MARTINS, Flavia L.; CASTRO, Paulo de C.; VENTURINI, Gabriela; SEGURO, Antonio C.; GIRARDI, Adriana C. C.
    Objectives: Clinical and epidemiological studies have suggested a correlation between vitamin D deficiency (VDD) and high blood pressure (BP). This study aimed to test the hypothesis that high BP induced by VDD is associated with altered expression and covalent modification of apical sodium transporters along the nephron. The contributions of the intrarenal renin-angiotensin system (RAS) and oxidative stress were also investigated. Methods: Male Wistar rats were fed a vitamin D-free (n = 26) or standard diet (n = 25) for 30 days. BP was recorded using noninvasive and invasive procedures. The expression levels of total and phosphorylated apical sodium transporters in rat renal cortex and medulla were evaluated by immunoblotting. Intrarenal RAS components were assessed by immunoblotting and ELISA. Renal oxidative stress was analyzed by measuring the concentrations of thiobarbituric acid reactive substances and reduced glutathione. Results: Higher BP levels in VDD rats than controls were accompanied by overexpression and hyperphosphorylation of renal cortical and medullary Na+-K+-2Cl- cotransporter type 2, enhanced levels of phosphorylated Na+/H+ exchanger type 3, and reduced expression levels of total and phosphorylated Na+/Cl- cotransporter. Changes in intrarenal RAS induced by VDD vs. controls included the marked elevation of medullary renin expression, higher expression of cortical angiotensinogen, higher urinary angiotensinogen excretion, and higher cortical and medullary angiotensin II content. VDD rats displayed higher thiobarbituric acid reactive substances/glutathione ratios in the renal cortex and medulla than controls. Conclusion: These results suggest that the molecular mechanisms underlying the effects of VDD on BP may include the upregulation of Na+-K+-2Cl- cotransporter type 2 and activation of intrarenal RAS and oxidative stress.