LUIZ FERNANDO ONUCHIC
Projetos de Pesquisa
Unidades Organizacionais
Departamento de Clínica Médica, Faculdade de Medicina - Docente
LIM/29 - Laboratório de Nefrologia Celular, Genética e Molecular, Hospital das Clínicas, Faculdade de Medicina - Líder
LIM/29 - Laboratório de Nefrologia Celular, Genética e Molecular, Hospital das Clínicas, Faculdade de Medicina - Líder
11 resultados
Resultados de Busca
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- Influence of androgenic blockade with flutamide on pain behaviour and expression of the genes that encode the NaV1.7 and NaV1.8 voltage-dependent sodium channels in a rat model of postoperative pain(2019) BARBOSA NETO, Jose Osvaldo; GARCIA, Joao Batista Santos; CARTAGENES, Maria do Socorro de Souza; AMARAL, Andressa Godoy; ONUCHIC, Luiz Fernando; ASHMAWI, Hazem AdelBackgroundExperimental studies suggest that testosterone reduces the nociceptive response after inflammatory and neuropathic stimuli, however the underlying mechanisms have not been fully elucidated. The aims of this study were to evaluate the effect of peripheral blockade of testosterone on pain behaviour and on expression levels of the genes that encode the NaV1.7 and NaV1.8 channels, in dorsal root ganglia in an acute postoperative pain model, as well as the influence of androgen blockade on the expression of these genes.MethodsPostoperative pain was induced by a plantar incision and the study group received flutamide to block testosterone receptor. The animals were submitted to behavioural evaluation preoperatively, 2h after incision, and on the 1st, 2nd, 3rd and 7th postoperative days. Von Frey test was used to evaluate paw withdrawal threshold after mechanical stimuli and the guarding pain test to assess spontaneous pain. The expression of the genes encoding the sodium channels at the dorsal root ganglia was determined by real time quantitative polymerase chain reaction.ResultsAnimals treated with flutamide presented lower paw withdrawal threshold at the 1st, 2nd, 3rd, and 7th postoperative days. The guarding pain test showed significant decrease in the flutamide group at 2h and on the 3rd and 7th postoperative days. No difference was detected between the study and control groups for the gene expression.ConclusionsOur data suggest an antinociceptive effect of androgens following plantar incision. The expression of genes that encode voltage-gated sodium channels was not influenced by androgen blockade.
- Disruption of polycystin-1 cleavage leads to cardiac metabolic rewiring in mice(2022) AMARAL, Andressa G.; SILVA, Camille C. C. da; SERNA, Julian D. C.; HONORATO-SAMPAIO, Kinulpe; FREITAS, Jessica A.; DUARTE-NETO, Amaro N.; BLOISE, Antonio C.; CASSINA, Laura; YOSHINAGA, Marcos Y.; CHAVES-FILHO, Adriano B.; QIAN, Feng; MIYAMOTO, Sayuri; BOLETTA, Alessandra; BORDIN, Silvana; KOWALTOWSKI, Alicia J.; ONUCHIC, Luiz F.Cardiovascular manifestations account for marked morbi-mortality in autosomal dominant polycystic kidney disease (ADPKD). Pkd1- and Pkd2-deficient mice develop cardiac dysfunction, however the underlying mechanisms remain largely unclear. It is unknown whether impairment of polycystin-1 cleavage at the G-proteincoupled receptor proteolysis site, a significant ADPKD mutational mechanism, is involved in this process. We analyzed the impact of polycystin-1 cleavage on heart metabolism using Pkd1V/V mice, a model unable to cleave this protein and with early cardiac dysfunction. Pkd1V/V hearts showed lower levels of glucose and amino acids and higher lipid levels than wild-types, as well as downregulation of p-AMPK, p-ACC beta, CPT1B-Cpt1b, Ppara, Nppa and Acta1. These findings suggested decreased fatty acid beta-oxidation, which was confirmed by lower oxygen consumption by Pkd1V/V isolated mitochondria using palmitoyl-CoA. Pkd1V/V hearts also presented increased oxygen consumption in response to glucose, suggesting that alternative substrates may be used to generate energy. Pkd1V/V hearts displayed a higher density of decreased-size mitochondria, a finding associated with lower MFN1, Parkin and BNIP3 expression. These derangements were correlated with increased apoptosis and inflammation but not hypertrophy. Notably, Pkd1V/V neonate cardiomyocytes also displayed shifts in oxygen consumption and p-AMPK downregulation, suggesting that, at least partially, the metabolic alterations are not induced by kidney dysfunction. Our findings reveal that disruption of polycystin-1 cleavage leads to cardiac metabolic rewiring in mice, expanding the understanding of heart dysfunction associated with Pkd1 deficiency and likely with human ADPKD.
- Cardiac dysfunction in Pkd1-deficient mice with phenotype rescue by galectin-3 knockout(2016) BALBO, Bruno E.; AMARAL, Andressa G.; FONSECA, Jonathan M.; CASTRO, Isac de; SALEMI, Vera M.; SOUZA, Leandro E.; SANTOS, Fernando dos; IRIGOYEN, Maria C.; QIAN, Feng; CHAMMAS, Roger; ONUCHIC, Luiz F.Alterations in myocardial wall texture stand out among ADPKD cardiovascular manifestations in hypertensive and normotensive patients. To elucidate their pathogenesis, we analyzed the cardiac phenotype in Pkd1(cond/cond) Nestin(cre) (CYG+) cystic mice exposed to increased blood pressure, at 5 to 6 and 20 to 24 weeks of age, and Pkd1(+/-) (HTG+) noncystic mice at 5-6 and 10-13 weeks. Echocardiographic analyses revealed decreased myocardial deformation and systolic function in CYG+ and HTG+ mice, as well as diastolic dysfunction in older CYG+ mice, compared to their Pkd1(cond/cond) and Pkd1(+/+) controls. Hearts from CYG+ and HTG+ mice presented reduced polycystin-1 expression, increased apoptosis, and mild fibrosis. Since galectin-3 has been associated with heart dysfunction, we studied it as a potential modifier of the ADPKD cardiac phenotype. Double-mutant Pkd1(cond/cond):Nestin(cre);Lgals(3-/-) (CYG-) and Pkd1(+/-);Lgals(3-/-) (HTG-) mice displayed improved cardiac deformability and systolic parameters compared to single -mutants, not differing from the controls. CYG- and HTG- showed decreased apoptosis and fibrosis. Analysis of a severe cystic model (Pkd1(v/v); VVG+) showed that Pkd1(v/v);Lgals(3-/-) (VVG-) mice have longer survival, decreased cardiac apoptosis and improved heart function compared to VVG+. CYG- and VVG- animals showed no difference in renal cystic burden compared to CYG+ and VVG+ mice. Thus, myocardial dysfunction occurs in different Pkdl-deficient models and suppression of galectin-3 expression rescues this phenotype.
- Smoking accelerates renal cystic disease and worsens cardiac phenotype in Pkd1-deficient mice(2021) SOUSA, Marciana V.; AMARAL, Andressa G.; FREITAS, Jessica A.; MURATA, Gilson M.; WATANABE, Elieser H.; BALBO, Bruno E.; TAVARES, Marcelo D.; HORTEGAL, Renato A.; ROCON, Camila; SOUZA, Leandro E.; IRIGOYEN, Maria C.; SALEMI, Vera M.; ONUCHIC, Luiz F.Smoking has been associated with renal disease progression in ADPKD but the underlying deleterious mechanisms and whether it specifically worsens the cardiac phenotype remain unknown. To investigate these matters, Pkd1-deficient cystic mice and noncystic littermates were exposed to smoking from conception to 18 weeks of age and, along with nonexposed controls, were analyzed at 13-18 weeks. Renal cystic index and cyst-lining cell proliferation were higher in cystic mice exposed to smoking than nonexposed cystic animals. Smoking increased serum urea nitrogen in cystic and noncystic mice and independently enhanced tubular cell proliferation and apoptosis. Smoking also increased renal fibrosis, however this effect was much higher in cystic than in noncystic animals. Pkd1 deficiency and smoking showed independent and additive effects on reducing renal levels of glutathione. Systolic function and several cardiac structural parameters were also negatively affected by smoking and the Pkd1-deficient status, following independent and additive patterns. Smoking did not increase, however, cardiac apoptosis or fibrosis in cystic and noncystic mice. Notably, smoking promoted a much higher reduction in body weight in Pkd1-deficient than in noncystic animals. Our findings show that smoking aggravated the renal and cardiac phenotypes of Pkd1-deficient cystic mice, suggesting that similar effects may occur in human ADPKD.
- Abdominal pain, arthritis, and nephrotic syndrome in a Syrian patient(2012) BALBO, Bruno Eduardo P.; SILVA, Andre Albuquerque; AMARAL, Andressa Godoy; MALHEIROS, Denise M. A. C.; ONUCHIC, Luiz Fernando; BARROS, Rui Toledo
- Ppia is the most stable housekeeping gene for qRT-PCR normalization in kidneys of three Pkd1-deficient mouse models(2021) MUNOZ, Juan J.; ANAUATE, Ana C.; AMARAL, Andressa G.; FERREIRA, Frederico M.; WATANABE, Elieser H.; MECA, Renata; ORMANJI, Milene S.; BOIM, Mirian A.; ONUCHIC, Luiz F.; HEILBERG, Ita P.Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common inherited renal disorder, characterized by renal cyst development leading to end-stage renal disease. Although the appropriate choice of suitable reference is critical for quantitative RNA analysis, no comparison of frequently used ""housekeeping"" genes is available. Here, we determined the validity of 7 candidate housekeeping genes (Actb, Actg1, B2m, Gapdh, Hprt, Pgam1 and Ppia) in kidney tissues from mouse models orthologous to ADPKD, including a cystic mice (CY) 10-12 weeks old (Pkd1(flox/flox):Nestin(cre)/Pkd1(flox/-):Nestin(cre), n = 10) and non-cystic (NC) controls (Pkd1(flox)/(flox)/Pkd1(flox)/(-), n = 10), Pkd1-haploinsufficient (HT) mice (Pkd1(+/-), n = 6) and wild-type (WT) controls (Pkd1(+/+), n = 6) and a severely cystic (SC) mice 15 days old (Pkd1(V/V), n = 7) and their controls (CO, n = 5). Gene expression data were analyzed using six distinct statistical softwares. The estimation of the ideal number of genes suggested the use of Ppia alone as sufficient, although not ideal, to analyze groups altogether. Actb, Hprt and Ppia expression profiles were correlated in all samples. Ppia was identified as the most stable housekeeping gene, while Gapdh was the least stable for all kidney samples. Stat3 expression level was consistent with upregulation in SC compared to CO when normalized by Ppia expression. In conclusion, present findings identified Ppia as the best housekeeping gene for CY + NC and SC + CO groups, while Hprt was the best for the HT + WT group.
- Identification of housekeeping genes for microRNA expression analysis in kidney tissues of Pkd1 deficient mouse models(2020) MUNOZ, J. J.; ANAUATE, A. C.; AMARAL, A. G.; FERREIRA, F. M.; MECA, R.; ORMANJI, M. S.; BOIM, M. A.; ONUCHIC, L. F.; HEILBERG, I. P.Polycystic kidney disease is a complex clinical entity which comprises a group of genetic diseases that leads to renal cyst development. We evaluated the most suitable housekeeping genes for microRNA expression by RT-qPCR analyses of kidney tissues in Pkd1-deficient mouse models from a panel of five candidates genes (miR-20a, miR-25, miR-26a, miR-191 and U6) and 3 target genes (miR-17, miR-21 and let-7a) using samples from kidneys of cystic mice (Pkd1(flox/flox):Nestin(cre), CY), non-cystic controls (Pkd1(flox/flox), NC), Pkd1-haploinsufficient (Pkd1(+/-), HT), wild-type controls (Pkd1(+/+), WT), severely cystic mice (Pkd1(V/V), SC), wild-type controls (CO). The stability of the candidate genes was investigated using NormFinder, GeNorm, BestKeeper, DataAssist, and RefFinder software packages and the comparative Delta Ct method. The analyses identified miR-26a as the most stable housekeeping gene for all kidney samples, miR-20a for CY and NC, miR-20a and miR-26a for HT and WT, and miR-25 and miR-26a for SC and CO. Expression of miR-21 was upregulated in SC compared to CO and trends of miR-21 upregulation and let-7a downregulation in CY and HT compared to its control kidneys, when normalized by different combinations of miR-20a, miR-25 and miR-26a. Our findings established miR-20a, miR-25, and miR-26a as the best housekeeping genes for miRNA expression analyses by RT-qPCR in kidney tissues of Pkd1-deficient mouse models.
- Periostin and polycystic kidney disease: more pieces in the puzzle(2019) WATANABE, Elieser H.; AMARAL, Andressa G.; ONUCHIC, Luiz F.
- Renal cyst growth is the main determinant for hypertension and concentrating deficit in Pkd1-deficient mice(2014) FONSECA, Jonathan M.; BASTOS, Ana P.; AMARAL, Andressa G.; SOUSA, Mauri F.; SOUZA, Leandro E.; MALHEIROS, Denise M.; PIONTEK, Klaus; IRIGOYEN, Maria C.; WATNICK, Terry J.; ONUCHIC, Luiz F.We have bred a Pkd1 floxed allele with a nestin-Cre expressing line to generate cystic mice with preserved glomerular filtration rate to address the pathogenesis of complex autosomal dominant polycystic kidney disease (ADPKD) phenotypes. Hypertension affects about 60% of these patients before loss of renal function, leading to significant morbimortality. Cystic mice were hypertensive at 5 and 13 weeks of age, a phenotype not seen in noncystic controls and Pkd1-haploinsufficient animals that do not develop renal cysts. Fractional sodium excretion was reduced in cystic mice at these ages. Angiotensinogen gene expression was higher in cystic than noncystic kidneys at 18 weeks, while ACE and the All receptor were expressed in renal cyst epithelia. Cystic animals displayed increased renal cAMP, cell proliferation, and apoptosis. At 24 weeks, mean arterial pressure and fractional sodium excretion did not significantly differ between the cystic and noncystic groups, whereas cardiac mass increased in cystic mice. Renal concentrating deficit is also an early finding in ADPKD. Maximum urine osmolality and urine nitrite excretion were reduced in 10-13- and 24-week-old cystic mice, deficits not found in haploinsufficient and noncystic controls. A trend of higher plasma vasopressin was observed in cystic mice. Thus, cyst growth most probably plays a central role in early-stage ADPKD-associated hypertension, with activation of the intrarenal renin-angiotensin system as a key mechanism. Cyst expansion is also likely essential for the development of the concentrating deficit in this disease. Our findings are consistent with areas of reduced perfusion in the kidneys of patients with ADPKD.
- Regulation of CFTR Expression and Arginine Vasopressin Activity Are Dependent on Polycystin-1 in Kidney-Derived Cells(2016) BARBOSA, Carolina Monteiro de Lemos; SOUZA-MENEZES, Jackson; AMARAL, Andressa Godoy; ONUCHIC, Luiz Fernando; CEBOTARU, Liudmila; GUGGINO, William B.; MORALES, Marcelo M.Background: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of multiple, progressive, fluid-filled renal cysts that distort the renal parenchyma, leading to end-stage renal failure, mainly after the fifth decade of life. ADPKD is caused by a mutation in the PKD1 or PKD2 genes that encode polycystin-1 (PC-1) and polycystin-2 (PC-2), respectively. PC-1 is an important regulator of several signaling pathways and PC-2 is a nonselective calcium channel. The CFTR chloride channel is responsible for driving net fluid secretion into the cysts, promoting cyst growth. Arginine vasopressin hormone (AVP), in turn, is capable of increasing cystic intracellular cAMP, contributing to cell proliferation, transepithelial fluid secretion, and therefore to disease progression. The aim of this study was to assess if AVP can modulate CFTR and whether PC-1 plays a role in this potential modulation. Methods: M1 cells, derived from mouse cortical collecting duct, were used in the current work. The cells were treated with 10(-7) M AVP hormone and divided into two main groups: transfected cells superexpressing PC-1 (Transf) and cells not transfected (Ctrl). CFTR expression was assessed by immunodetection, CFTR mRNA levels were quantified by quantitative reverse transcription-polymerase chain reaction, and CFTR net ion transport was measured using the Ussing chamber technique. Results: AVP treatment increased the levels of CFTR protein and mRNA. CFTR short-circuit currents were also increased. However, when PC-1 was overexpressed in M1 cells, no increase in any of these parameters was detected. Conclusions: CFTR chloride channel expression is increased by AVP in M1 cells and PC-1 is capable of regulating this modulation.