MARIA HELOISA MASSOLA SHIMIZU
Projetos de Pesquisa
Unidades Organizacionais
Departamento de Clínica Médica, Faculdade de Medicina
LIM/12 - Laboratório de Pesquisa Básica em Doenças Renais, Hospital das Clínicas, Faculdade de Medicina
LIM/12 - Laboratório de Pesquisa Básica em Doenças Renais, Hospital das Clínicas, Faculdade de Medicina
8 resultados
Resultados de Busca
Agora exibindo 1 - 8 de 8
- Vitamin D Deficiency Aggravates Chronic Kidney Disease Progression after Ischemic Acute Kidney Injury(2014) GONCALVES, Janaina Garcia; BRAGANCA, Ana Carolina de; CANALE, Daniele; SHIMIZU, Maria Heloisa Massola; SANCHES, Talita Rojas; MOYSES, Rosa Maria Affonso; ANDRADE, Lucia; SEGURO, Antonio Carlos; VOLPINI, Rildo AparecidoBackground: Despite a significant improvement in the management of chronic kidney disease (CKD), its incidence and prevalence has been increasing over the years. Progressive renal fibrosis is present in CKD and involves the participation of several cytokines, including Transforming growth factor-beta 1 (TGF-beta 1). Besides cardiovascular diseases and infections, several studies show that Vitamin D status has been considered as a non-traditional risk factor for the progression of CKD. Given the importance of vitamin D in the maintenance of essential physiological functions, we studied the events involved in the chronic kidney disease progression in rats submitted to ischemia/reperfusion injury under vitamin D deficiency (VDD). Methods: Rats were randomized into four groups: Control; VDD; ischemia/reperfusion injury (IRI); and VDD+IRI. At the 62 day after sham or IRI surgery, we measured inulin clearance, biochemical variables and hemodynamic parameters. In kidney tissue, we performed immunoblotting to quantify expression of Klotho, TGF-beta, and vitamin D receptor (VDR); gene expression to evaluate renin, angiotensinogen, and angiotensin-converting enzyme; and immunohistochemical staining for ED1 (macrophages), type IV collagen, fibronectin, vimentin, and alpha-smooth mucle actin. Histomorphometric studies were performed to evaluate fractional interstitial area. Results: IRI animals presented renal hypertrophy, increased levels of mean blood pressure and plasma PTH. Furthermore, expansion of the interstitial area, increased infiltration of ED1 cells, increased expression of collagen IV, fibronectin, vimentin and alpha-actin, and reduced expression of Klotho protein were observed. VDD deficiency contributed to increased levels of plasma PTH as well as for important chronic tubulointerstitial changes (fibrosis, inflammatory infiltration, tubular dilation and atrophy), increased expression of TGF-beta 1 and decreased expression of VDR and Klotho protein observed in VDD+IRI animals. Conclusion: Through inflammatory pathways and involvement of TGF-beta 1 growth factor, VDD could be considered as an aggravating factor for tubulointerstitial damage and fibrosis progression following acute kidney injury induced by ischemia/reperfusion.
- Vitamin D Deficiency Aggravates the Renal Features of Moderate Chronic Kidney Disease in 5/6 Nephrectomized Rats(2018) BRAGANCA, Ana Carolina de; CANALE, Daniele; GONCALVES, Janaina Garcia; SHIMIZU, Maria Heloisa Massola; SEGURO, Antonio Carlos; VOLPINI, Rildo AparecidoThe pathogenesis of chronic kidney disease (CKD) involves a very complex interaction between hemodynamic and inflammatory processes, leading to glomerular/vascular sclerosis, and fibrosis formation with subsequent evolution to end-stage of renal disease. Despite efforts to minimize the progression of CKD, its incidence and prevalence continue to increase. Besides cardiovascular diseases and infections, several studies demonstrate that vitamin D status could be considered as a non-traditional risk factor for the progression of CKD. Therefore, we investigated the effects of vitamin D deficiency (VDD) in the course of moderate CKD in 5/6 nephrectomized rats (Nx). Adult male Wistar rats underwent Sham surgery or Nx and were subdivided into the following four groups: Sham, receiving standard diet (Sham); Sham VDD, receiving vitamin D-free diet (VDD); Nx, receiving standard diet (Nx); and VDD+Nx, receiving vitamin D-free diet (VDD+Nx). Sham or Nx surgeries were performed 30 days after standard or vitamin D-free diets administration. After validation of vitamin D depletion, we considered only Nx and VDD+Nx groups for the following studies. Sixty days after surgeries, VDD+Nx rats exhibited hypertension, a greater decline in renal function and plasma FGF-23 levels, renal hypertrophy, as well as higher plasma levels of PTH and aldosterone. In addition, those animals presented more significant chronic tubulointerstitial changes (cortical interstitial expansion/inflammation/fibrosis), higher expression of collagen IV, fibronectin and alpha-smooth muscle actin, and lower expressions of JG12 and M2 macrophages. Also, VDD+Nx rats had greater infiltration of inflammatory cells (M1 macrophages and T-cells). Such changes were accompanied by higher expression of TGF-beta 1 and angiotensinogen and decreased expression of VDR and Klotho protein. Our observations indicate that vitamin D deficiency impairs the renal function and worsens the renovascular and morphological changes, aggravating the features of moderate CKD in 5/6 nephrectomized rats.
- Vitamin D deficiency aggravates ischemic acute kidney injury in rats(2015) BRAGANCA, Ana Carolina de; VOLPINI, Rildo A.; CANALE, Daniele; GONCALVES, Janaina G.; SHIMIZU, Maria Heloisa M.; SANCHES, Talita R.; SEGURO, Antonio C.; ANDRADE, LuciaVitamin D deficiency (VDD) increases the risk of death in hospitalized patients. Renal ischemia/reperfusion injury (IRI) induces acute kidney injury (AKI), which activates cell cycle inhibitors, including p21, a cyclindependent kinase inhibitor and genomic target of 25-hydroxyvitamin D, which is in turn a potent immunomodulator with antiproliferative effects. In this study, we assess the impact of VDD in renal IRI. Wistar rats were divided into groups, each evaluated for 30 days: control (receiving a standard diet); VDD (receiving a vitamin D-free diet); IRI (receiving a standard diet and subjected to 45-min bilateral renal ischemia on day 28); and VDD + IRI (receiving a vitamin D-free diet and subjected to 45-min bilateral renal ischemia on day 28). At 48 h after IRI, animals were euthanized; blood, urine, and kidney tissue samples were collected. Compared with IRI rats, VDD + IRI rats showed a more severe decrease in glomerular filtration rate, greater urinary protein excretion, a higher kidney/body weight ratio and lower renal aquaporin 2 expression, as well as greater morphological damage, characterized by increased interstitial area and tubular necrosis. Our results suggest that the severity of tubular damage in IRI may be associated with downregulation of vitamin D receptors and p21. VDD increases renal inflammation, cell proliferation and cell injury in ischemic AKI.
conferenceObject VITAMIN D DEFICIENCY AGGRAVATES TENOFOVIR INDUCED METABOLIC SYNDROME AND RENAL FAILURE(2013) CANALE, Daniele; BRAGANCA, Ana Carolina de; GONCALVES, Janana; SHIMIZU, Maria Helosa M.; VOLPINI, Rildo A.; ANDRADE, Lucia; SEGURO, Antonio CarlosIntroduction and Aims:Vitamin D deficiency (VDD) is highly prevalent among HIV-infected individuals. Tenofovir disoproxil fumarate (TDF), a widely used component of antiretroviral regimens for HIV treatment, has been associated with comorbidities, some of which have been attributed to renal toxicity and metabolic syndrome. The aim of the study was to investigate the effect of VDD on TDF treated rats. Methods:Wistar rats were randomly divided into four groups: control (C, n = 9), receiving a standard diet for 60 days; VDD (n = 6), receiving a free-vitamin D diet for 60 days; TDF (n = 9), receiving a standard diet for 60 days with the addition of TDF (50 mg/kg food) for the last 30 days; and VDD+TDF (n = 7) received a free-vitamin D diet for 60 days with the addition of TDF (50 mg/kg food) for the last 30 days. We measured inulin clearance (GFR, mL/min/100g); blood pressure (BP, mmHg), renal blood flow and calculated renal vascular resistance (RVR, mmHg/mL/min); serum levels of 25-hydroxyvitamin D (25(OH)D, ng/mL), cholesterol (mg/dL) and triglycerides (mg/dL); urinary sodium excretion (UVNa,mEq/24h). In renal tissue, we immunoblotted for angiotensin II (AII), endothelial nitric oxide synthase (eNOS) and vitamin D receptor (VDR). Data are expressed as mean ± SEM. Results:Vitamin D levels were similar in C (15.4±1 ng/mL) and TDF (14.8±1.3 ng/mL) groups and <1.5 ng/mL in VDD groups. Body weight, water intake and food ingestion were not different among the 4 groups. Treatment with TDF led to impaired renal function, hypertension, higher RVR and dyslipidemia. Administration of TDF also increased protein expression of AII and VDR. Association of TDF and VDD exacerbates TDF nephrotoxicity, as well as metabolic syndrome. Furthermore, VDD+TDF group showed urinary sodium retention. The increased VDR protein expression in TDF groups may represent a compensatory effect to decrease renal injury. Conclusions:VDD aggravates renovascular effects and TDF-induced renal failure at least in part due to the involvement of renin-angiotensin-aldosterone system. Therefore, the assessment of vitamin D is important in HIV patients receiving TDF.conferenceObject VITAMIN D DEFICIENCY IS ASSOCIATED WITH DOWNREGULATION OF KLOTHO AND DEVELOPMENT OF FIBROSIS IN A MURINE MODEL OF RENAL ISCHEMIA/REPERFUSION INJURY(2013) GONCALVES, Janaina G.; CANALE, Daniele; BRAGANCA, Ana Carolina de; SHIMIZU, Maria Heloisa M.; MOYSES, Rosa Maria A.; ANDRADE, Lucia; SEGURO, Antonio C.; VOLPINI, Rildo A.Introduction and Aims:Vitamin D deficiency (VDD) is highly prevalent in chronic kidney disease (CKD) patients. Ischemia/reperfusion-Acute Kidney Injury (IR-AKI) is considered a risk factor for CKD progression. Previous studies suggests that activation of the renin-angiotensin-aldosterone system (RAAS) and VDD could be involved in reduction of Klotho expression, an early marker of stage 1 CKD. The aim of this study was to investigate the effect of VDD on klotho expression and fibrosis development in a model of IR-AKI. Methods:Male Wistar rats (180-200g) were randomly divided into four groups (n=8 each): control (C) and ischemic (IR) fed a standard diet; VDD and VDD+IR, fed a free-vitamin D diet. On day 28, IR and VDD+IR rats were submitted to 45-minute clamping of both renal arteries. On day 90, we measured inulin clearance (GFR); Mean arterial blood pressure (MAP), renal blood flow (RBF) and calculated renal vascular resistance (RVR). We also measured serum levels of 25-hydroxyvitamin D (25(OH)D, ng/mL) and proteinuria. In renal tissue, we immunoblotted for klotho and performed imunohistochemical assay for collagen IV and fibronectin. We estimated fibrosis by fractional interstitial area (FIA) and fibronectin/collagen IV expression by score ranging from 0 to 4 according to the extension of staining. Data are expressed as mean ± SEM. Table 1. Hemodynamic and biochemical data and fractional interstitial area, Western Blot and Immunohistochemical studies. Results:Vitamin D levels were similar in C (15.4±1) and IR (15±0.6) groups and <1.5 ng/mL in VDD groups. GFR, RBF and RVR were not different among the studied groups. MAP was markedly elevated in VDD, IR and VDD+IR groups, reflecting a possible interaction on RAAS. Also, VDD, IR and VDD+IR groups showed larger areas of fibrosis and higher scores for fibronectin and collagen IV and decreased levels of klotho. Conclusions:The increased proteinuria/fibrosis and fibronectin/collagen IV expression in VDD+IR rats suggests progressive renal injury. Downregulation of klotho expression is a possible marker of chronification. Our study shows a very plausible role of VDD in this pathway as a potential stimulus for fibrosis.- Vitamin D Deficiency Aggravates Nephrotoxicity, Hypertension and Dyslipidemia Caused by Tenofovir: Role of Oxidative Stress and Renin-Angiotensin System(2014) CANALE, Daniele; BRAGANCA, Ana Carolina de; GONCALVES, Janaina Garcia; SHIMIZU, Maria Heloisa Massola; SANCHES, Talita Rojas; ANDRADE, Lucia; VOLPINI, Rildo Aparecido; SEGURO, Antonio CarlosVitamin D deficiency (VDD) is prevalent among HIV-infected individuals. Vitamin D has been associated with renal and cardiovascular diseases because of its effects on oxidative stress, lipid metabolism and renin-angiotensin-aldosterone system (RAAS). Tenofovir disoproxil fumarate (TDF), a widely used component of antiretroviral regimens for HIV treatment, can induce renal injury. The aim of this study was to investigate the effects of VDD on TDF-induced nephrotoxicity. Wistar rats were divided into four groups: control, receiving a standard diet for 60 days; VDD, receiving a vitamin D-free diet for 60 days; TDF, receiving a standard diet for 60 days with the addition of TDF (50 mg/kg food) for the last 30 days; and VDD+ TDF receiving a vitamin D-free diet for 60 days with the addition of TDF for the last 30 days. TDF led to impaired renal function, hyperphosphaturia, hypophosphatemia, hypertension and increased renal vascular resistance due to downregulation of the sodium-phosphorus cotransporter and upregulation of angiotensin II and AT1 receptor. TDF also increased oxidative stress, as evidenced by higher TBARS and lower GSH levels, and induced dyslipidemia. Association of TDF and VDD aggravated renovascular effects and TDF-induced nephrotoxicity due to changes in the redox state and involvement of RAAS.
conferenceObject N-ACETYLCYSTEINE ATTENUATES RENAL AND LUNG ALTERATIONS INDUCED BY SENESCENCE IN THE RAT(2012) VOLPINI, Rildo Aparecido; BRAGANCA, Ana Carolina De; CANALE, Daniele; GONCALVES, Janaina Garcia; BRANDAO, Thais Prevital Bastos; ANDRADE, Lucia; SEGURO, Antonio Carlos; SHIMIZU, Maria Heloisa MassolaObjectives: Clinical flow cytometric analysis of neoplastic hematolymphoid cells relies on evaluation of surface membrane and intracellular antigens expression patterns, which are related to normal cells of a particular lineage and maturational stage. A complete characterization of the studied population includes information about presence, absence and level of expression of a series of relevant antigens recognized by highly specific monoclonal antibodies (MAbs). Usually, the fluorescence intensity is considered to be directly proportional to the amount of antigen expressed in a specific population, as assessed by flow cytometry using the mean peak of channel fluorescence (MPCF), the proper use of MAbs is essential for this condition to be valid. Data show that laboratories use a wide variety of MAb dilutions, even those obtained from the same manufacturer. Ideally, the titre value should be determined for every MAb and is defined as the amount of antibody that is required to satura te the maximum number of antigen binding sites on a selected cell population, ensuring that antibodies are used efficiently yet still remain in excess. Our objective is to report a new statistical criteria adopted by our laboratory to establish optimal MAb dilution for flow cytometry analysis based on the discrimination of unstained and positively stained cells using MPCF. Methods: We have tested in our laboratory 57 MAbs from different manufacturers, from May/2010 until August/2011 (27 conjugated to FITC, 11 to PE, 4 to PerCP/PEDY647/CyQ or RPECy5 and 5 to APC). The tests were performed using bone marrow or peripheral blood samples which presented any positive population (normal or neoplastic) for the studied MAbs. The staining procedure followed manufacturers ́ protocols, with different volumes of Mabs (recommended by manufacturer followed by titrations 1:2, 1:4 and >1:4). At least 10,000 events were acquired for each sample using the flow cytometer BD FACSCalliburTM, and data was analyzed by CellQuestTM, obtaining the MPCF values for unstained and positively stained cells. The percentage difference between the MPCF (PD-MPCF) values was calculated applying this formula: (MPCF from positively stained population–MPCF from unstained population) x 100/MPCF from positively stained population. An adequate PD-MPCF must be 95% or more. Results: Among MAbs tested, 42/57 (73,68%) were likely to be titrated, 18 being titrated to 1:2, 22 to 1:4 and 2 to >1:4. Analysis according to the fluorochrome conjugated to MAb demonstrated more efficient titration for APC (3 MAbs in 5), while those conjugated with FITC showed difficulty in titration (9/27 have not shown good performance at lower concentrations than those indicated by the manufacturer and titers >1:4 were not appropriate for any samples). Six MAbs had titration disapproved by the criterion used (PD-MPCF) but were also disapproved in the concentration recommended by the manufacturer because the expected fluorescence level was dim for the population analyzed. Conclusions: We conclude that PD-MPCF is an efficient and objective instrument as quality control for validation and titration of MAbs, and that the cutoff of 95% ensures proper separation between positive and negative stained populations showing good correlation with the performance in daily routine.Introduction and Aims: Aging is defined as a deterioration of the physiological functions necessary for survival. Oxidative stress increases with age and is associated with alterations in kidney and lung function. Previous studies have demonstrated that N-acetylcysteine (NAC), an antioxidant, protects animals from kidney and lung injury. The aim of this study was to evaluate the effects of NAC on sodium and water transport in the kidneys and lungs of aging rats. Methods: Normal 8-month-old male Wistar rats were treated or not (n = 6 per group) with NAC (600 mg/L in drinking water) and followed for 16 months. At the end of the follow-up period (i.e., in 24-month-old rats), we measured thiobarbituric acid reactive substances (TBARS), a marker of lipid peroxidation, urine volume (UV), fractional excretion of water (FE H2O) and inulin clearance (CIn). In addition, we performed immunoblotting for proteins in the kidneys—for the Na-K-2Cl cotransporter NKCC2, the alpha subunit of the epithelial sodium channel (a-ENaC), aquaporin 2 (AQP2), and the urea transporter UT-A1—and in the lungs—for a-ENaC, the Na-K-2Cl cotransporter NKCC1 and AQP5. Using immunohistochemistry, we evaluated the amount of ED1-positive cells (macrophages/monocytes) per grid field (0.087 mm2) in renal tissue samples. Results: Mean NAC ingestion was 24 ± 2 mg/day. As shown in the table, NAC decreased serum TBARS, FE H2O and ameliorated CIn in aged rats. The renal expression of NKCC2, AQP2, and UT-A1 was significantly higher (69%, 42%, and 89% higher, respectively) in aged+NAC rats than in untreated aged rats, although that of a- ENaC was unaffected. Pulmonary expression of a-ENaC and AQP5 was 32% and 30% higher, respectively, in aged+NAC rats than in aged rats, whereas that of NKCC1 was 46% lower. In addition, there was significantly less infiltration by ED1-positive cells in the aged+NAC rats. Conclusions: In aged rats, NAC treatment ameliorated renal function and upregulated renal expression of NKCC2, AQP2, and UT-A1. These effects appear to increase urinary concentrating ability and dilution capacity in aged rats, thereby decreasing the incidence of hyper- or hypo- osmolarity in this population. In addition, NAC treatment reduced the infiltration of inflammatory cells. We also found that NAC profoundly influences sodium and water transport in alveolar epithelial cells, an effect that could decrease the incidence of lung edema in the elderly.- The Blockade of TACE-Dependent EGF Receptor Activation by Losartan-Erlotinib Combination Attenuates Renal Fibrosis Formation in 5/6-Nephrectomized Rats Under Vitamin D Deficiency(2021) GONCALVES, Janaina Garcia; CANALE, Daniele; BRAGANCA, Ana Carolina de; SEGURO, Antonio Carlos; SHIMIZU, Maria Heloisa Massola; VOLPINI, Rildo AparecidoChronic kidney disease (CKD) has been considered a major public health issue. In addition to cardiovascular diseases and infections, hypovitaminosis D has been considered a non-traditional aggravating factor for CKD progression. Interstitial fibrosis is a hallmark of CKD strongly correlated with deterioration of renal function. Transforming growth factor beta (TGF-beta) is the major regulatory profibrotic cytokine in CKD. Many injurious stimuli converge on the TGF-beta pathway, which has context-dependent pleiotropic effects and interacts with several related renal fibrosis formation (RFF) pathways. Epidermal growth factor receptor (EGFR) is critically involved in CKD progression, exerting a pathogenic role in RFF associated with TGF-beta-related fibrogenesis. Among others, EGFR pathway can be activated by a disintegrin and a metalloproteinase known as tumor necrosis factor alpha-converting enzyme (TACE). Currently no effective therapy is available to completely arrest RFF and slow the progression of CKD. Therefore, we investigated the effects of a double treatment with losartan potassium (L), an AT1R antagonist, and the tyrosine kinase inhibitor erlotinib (E) on the alternative pathway of RFF related to TACE-dependent EGFR activation in 5/6-nephrectomized rats under vitamin D deficiency (D). During the 90-day protocol, male Wistar rats under D, were submitted to 5/6 nephrectomy (N) on day 30 and randomized into four groups: N+D, no treatment; N+D+L, received losartan (50 mg/kg/day); N+D+E, received erlotinib (6 mg/kg/day); N+D+L+E received losartan+erlotinib treatment. N+D+L+E data demonstrated that the double treatment with losartan+erlotinib not only blocked the TACE-dependent EGF receptor activation but also prevented the expression of TGF-beta, protecting against RFF. This renoprotection by losartan+erlotinib was corroborated by a lower expression of ECM proteins and markers of phenotypic alteration as well as a lesser inflammatory cell infiltrate. Although erlotinib alone has been emerging as a renoprotective drug, its association with losartan should be considered as a potential therapeutic strategy on the modulation of RFF.