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1.
Int J Mol Sci ; 25(17)2024 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-39273100

RESUMO

Autophagy is the primary intracellular degradation system, and it plays an important role in many biological and pathological processes. Studies of autophagy involvement in developmental processes are important for understanding various processes. Among them are fibrosis, degenerative diseases, cancer development, and metastasis formation. Diabetic kidney disease is one of the main causes of chronic kidney disease and end-stage renal failure. The aim of this study was to investigate the immunohistochemical expression patterns of LC3B, LAMP2A, and GRP78 during different developmental stages of early-developing human kidneys and in samples from patients with type II diabetes mellitus. During the 7/8th DW, moderate expression of LC3B and LAMP2A and strong expression of GRP78 were found in the mesonephric glomeruli and tubules. In the 9/10th DW, the expression of LC3B and LAMP2A was even more pronounced in the mesonephric tubules. LC3B, LAMP2A, and GRP78 immunoreactivity was also found in the paramesonephric and mesonephric ducts and was stronger in the 9/10th DW compared with the 7/8th DW. In addition, the expression of LC3B, LAMP2A, and GRP78 also appeared in the mesenchyme surrounding the paramesonephric duct in the 9/10th DW. In the 15/16th DW, the expression of LC3B in the glomeruli was weak, that of LAMP2A was moderate, and that of GRP78 was strong. In the tubuli, the expression of LC3B was moderate, while the expression of LAMP2A and GRP78 was strong. The strongest expression of LC3B, LAMP2A, and GRP78 was observed in the renal medullary structures, including developing blood vessels. In postnatal human kidneys, the most extensive LC3B, LAMP2A, and GRP78 expression in the cortex was found in the epithelium of the proximal convoluted tubules, with weak to moderate expression in the glomeruli. The medullary expression of LC3B was weak, but the expression of LAMP2A and GRP78 was the strongest in the medullary tubular structures. Significantly lower expression of LC3B was found in the glomeruli of the diabetic patients in comparison with the nondiabetic patients, but there was no difference in the expression of LC3B in the tubule-interstitial compartment. The expression of LAMP2A was significantly higher in the tubule-interstitial compartments of the diabetic patients in comparison with the nondiabetic patients, while its expression did not differ in the glomeruli. Extensive expression of GRP78 was found in the glomeruli and the tubule-interstitial compartments, but there was no difference in the expression between the two groups of patients. These data give us new information about the expression of LC3B, LAMP2A, and GRP78 during embryonic, fetal, and early postnatal development. The spatiotemporal expression of LC3B, LAMP2A, and GRP78 indicates the important role of autophagy during the early stages of renal development. In addition, our data suggest a disturbance in autophagy processes in the glomeruli and tubuli of diabetic kidneys as an important factor in the pathogenesis of diabetic kidney disease.


Assuntos
Autofagia , Nefropatias Diabéticas , Chaperona BiP do Retículo Endoplasmático , Rim , Proteína 2 de Membrana Associada ao Lisossomo , Proteínas Associadas aos Microtúbulos , Humanos , Chaperona BiP do Retículo Endoplasmático/metabolismo , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/genética , Rim/metabolismo , Rim/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Biomarcadores/metabolismo , Feminino , Masculino , Proteínas de Choque Térmico/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia
2.
Int J Mol Sci ; 25(8)2024 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-38674142

RESUMO

The gradual deterioration of articular cartilage was thought to be the central event in osteoarthritis (OA), but recent studies demonstrated the importance of low-grade synovitis in the progression of OA. The Syndecan (SDC) family of membrane proteoglycans is known to be involved in the regulation of inflammation, but there is limited evidence considering the role of syndecans in OA synovitis. Our study aimed to investigate the hip OA synovial membrane expression patterns of SDC1, SDC2 and SDC4, as well as exostosins and sulfotransferases (enzymes involved in the polymerisation and modification of syndecans' heparan sulphate chains). Synovial membrane samples of patients with OA (24) were divided into two groups according to their Krenn synovitis score severity. The immunohistochemical expressions of SDC1, SDC2, SDC4, EXT1, EXT2, NDST1 and NDST2 in synovial intima and subintima were then analysed and compared with the control group (patients with femoral neck fracture). According to our study, the immunoexpression of SDC1, NDST1 and EXT2 is significantly increased in the intimal cells of OA synovial membrane in patients with lower histological synovitis scores and SDC4 in patients with higher synovitis scores, in comparison with non-OA controls. The difference in the expression of SDC2 among the OA and non-OA groups was insignificant. SDC1, SDC4, NDST1 and EXT2 seem to be involved as inflammation moderators in low-grade OA synovitis and, therefore, should be further investigated as potential markers of disease progression and therapeutic goals.


Assuntos
Biomarcadores , Osteoartrite do Quadril , Sulfotransferases , Sindecanas , Sinovite , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inflamação/metabolismo , Inflamação/patologia , N-Acetilglucosaminiltransferases , Osteoartrite do Quadril/metabolismo , Osteoartrite do Quadril/patologia , Sulfotransferases/metabolismo , Sindecanas/metabolismo , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Sinovite/metabolismo , Sinovite/patologia , Biomarcadores/análise
3.
Int J Mol Sci ; 25(13)2024 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-38999938

RESUMO

The purpose of this study was to evaluate the spatiotemporal immunoexpression pattern of microtubule-associated protein 1 light chain 3 beta (LC3B), glucose-regulated protein 78 (GRP78), heat shock protein 70 (HSP70), and lysosomal-associated membrane protein 2A (LAMP2A) in normal human fetal kidney development (CTRL) and kidneys affected with congenital anomalies of the kidney and urinary tract (CAKUT). Human fetal kidneys (control, horseshoe, dysplastic, duplex, and hypoplastic) from the 18th to the 38th developmental week underwent epifluorescence microscopy analysis after being stained with antibodies. Immunoreactivity was quantified in various kidney structures, and expression dynamics were examined using linear and nonlinear regression modeling. The punctate expression of LC3B was observed mainly in tubules and glomerular cells, with dysplastic kidneys displaying distinct staining patterns. In the control group's glomeruli, LAMP2A showed a sporadic, punctate signal; in contrast to other phenotypes, duplex kidneys showed significantly stronger expression in convoluted tubules. GRP78 had a weaker expression in CAKUT kidneys, especially hypoplastic ones, while normal kidneys exhibited punctate staining of convoluted tubules and glomeruli. HSP70 staining varied among phenotypes, with dysplastic and hypoplastic kidneys exhibiting stronger staining compared to controls. Expression dynamics varied among observed autophagy markers and phenotypes, indicating their potential roles in normal and dysfunctional kidney development.


Assuntos
Autofagia , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico HSP70 , Rim , Proteína 2 de Membrana Associada ao Lisossomo , Proteínas Associadas aos Microtúbulos , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/genética , Rim/metabolismo , Rim/anormalidades , Rim/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Anormalidades Urogenitais/metabolismo , Anormalidades Urogenitais/patologia , Sistema Urinário/metabolismo , Sistema Urinário/anormalidades , Refluxo Vesicoureteral/metabolismo , Refluxo Vesicoureteral/patologia
4.
Molecules ; 29(14)2024 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-39064873

RESUMO

Kidney failures in infants are mostly caused by congenital anomalies of the kidney and urinary tract (CAKUT), which are among the most common congenital birth disorders worldwide when paired with cardiac abnormalities. People with CAKUT often have severe kidney failure as a result of a wide range of abnormalities that can occur alone or in conjunction with other syndromic disorders. In this study, we aimed to investigate the expression pattern of CAKUT candidate genes alpha-8 integrin (ITGA8) and Van Gogh-like 2 (VANGL2) in fetal tissues of healthy and CAKUT-affected kidneys using immunohistochemistry and immunofluorescence. We found that under CAKUT circumstances, the expressions of ITGA8 and VANGL2 are changed. Additionally, we showed that VANGL2 expression is constant during fetal aging, but ITGA8 expression varies. Moreover, compared to normal healthy kidneys (CTRL), ITGA8 is poorly expressed in duplex kidneys (DKs) and dysplastic kidneys (DYS), whereas VANGL2 is substantially expressed in dysplastic kidneys (DYS) and poorly expressed in hypoplastic kidneys (HYP). These results point to VANGL2 and ITGA8 as potential prognostic indicators for CAKUT malformations. Further research is necessary to explore the molecular mechanisms underlying this differential expression of ITGA8 and VANGL2.


Assuntos
Cadeias alfa de Integrinas , Rim , Feminino , Humanos , Masculino , Cadeias alfa de Integrinas/metabolismo , Cadeias alfa de Integrinas/genética , Rim/metabolismo , Rim/anormalidades , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Sistema Urinário/metabolismo , Sistema Urinário/anormalidades , Anormalidades Urogenitais/metabolismo , Anormalidades Urogenitais/genética , Refluxo Vesicoureteral/metabolismo , Refluxo Vesicoureteral/genética
5.
Medicina (Kaunas) ; 60(8)2024 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-39202484

RESUMO

Background and Objectives: Colorectal cancer (CRC) is the most frequently diagnosed malignant disease of the gastrointestinal system, and new diagnostic and prognostic markers are needed to elucidate the complete tumor profile. Materials and Methods: We used CRC tumor tissues (Dukes' A-D) and adjacent noncancerous tissues of 43 patients. Immunohistochemistry was used to examine the expression of phosphodiesterase 4B (PDE4B), phosphodiesterase 4D (PDE4D), and secreted frizzled related protein 5 (SFRP5) markers. We also analyzed the expression levels of PDE4B, PDE4D, and SFRP5 in CRC tissues compared to control tissues using RNA-sequencing data from the UCSC Xena browser. Results: In CRC stages, the distribution of PDE4B-positive cells varied, with differing percentages between epithelium and lamina propria. Statistically significant differences were found in the number of PDE4B-positive epithelial cells between healthy controls and all CRC stages, as well as between different CRC stages. Similarly, significant differences were observed in the number of PDE4B-positive cells in the lamina propria between healthy controls and all CRC stages, as well as between different CRC stages. CRC stage Dukes' C exhibited a significantly higher number of PDE4B-positive cells in the lamina propria compared to CRC stage Dukes' B. Significant differences were noted in the number of PDE4D-positive epithelial cells between healthy controls and CRC stages Dukes' A, B, and D, as well as between CRC stage Dukes' C and stages A, B, and D. CRC stage Dukes' A had significantly more PDE4D-positive cells in the lamina propria compared to stage D. Significant differences were also observed in the number of SFRP5-positive cells in the lamina propria between healthy controls and all CRC stages, as well as between CRC stages Dukes' A and D. While the expression of PDE4D varied across CRC stages, the expression of SFRP5 remained consistently strong in both epithelium and lamina propria, with significant differences noted mainly in the lamina propria. The expression levels of PDE4B, PDE4D, and SFRP5 reveal significant differences in the expression of these genes between CRC patients and healthy controls, with notable implications for patient prognosis. Namely, our results demonstrate that PDE4B, PDE4D, and SFRP5 are significantly under-expressed in CRC tissues compared to control tissues. The Kaplan-Meier survival analysis and the log-rank (Mantel-Cox) test revealed distinct prognostic implications where patients with lower expression levels of SFRP5 exhibited significantly longer overall survival. The data align with our immunohistochemical results and might suggest a potential tumor-suppressive role for these genes in CRC. Conclusions: Considering significantly lower gene expression, aligned with our immunohistochemical data in tumor tissue in comparison to the control tissue, as well as the significantly poorer survival rate in the case of its higher expression, we can hypothesize that SFRP5 is the most promising biomarker for CRC out of the observed proteins. These findings suggest alterations in PDE4B, PDE4D, and SFRP5 expression during CRC progression, as well as between different stages of CRC, with potential implications for understanding the molecular mechanisms involved in CRC development and progression.


Assuntos
Biomarcadores Tumorais , Neoplasias Colorretais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Humanos , Neoplasias Colorretais/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Masculino , Feminino , Pessoa de Meia-Idade , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Idoso , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Imuno-Histoquímica/métodos , Adulto , Proteínas de Membrana/análise , Proteínas de Membrana/genética
6.
Int J Mol Sci ; 23(4)2022 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-35216141

RESUMO

This study aimed to explore how Dab1 gene functional silencing influences the spatial and temporal expression patterns of fibroblast growth factor receptor 1 (FGFR1), fibroblast growth factor receptor 2 (FGFR2), receptor-interacting protein kinase 5 (RIP5), and huntingtin-interacting protein 2 (HIP2) in the developing and postnatal kidneys of the yotari mice as potential determinants of normal kidney formation and function. Dab1-/- animal kidneys exhibit diminished FGFR1/FGFR2 expression in all examined developmental stages, whereas RIP5 cell immunoreactivity demonstrated negligible variation. The HIP2 expression revealed a discernible difference during the postnatal period, where we noted a significant decrease in almost all the observed kidney structures of yotari animals. An extracellular signal-regulated kinase (Erk1/2) and mammalian target of rapamycin (mTOR) expression in yotari kidneys decreased in embryonic and postnatal developmental phases for which we can hypothesize that the Erk1/2 signaling pathway in the yotari mice kidneys is dependent on Reelin with Dab1 only partially implicated in Reelin-mediated MEK/Erk1/2 activation. The impairment of FGFR1 and FGFR2 expression suggests the involvement of the observed markers in generating the CAKUT phenotype resulting in renal hypoplasia. Our study demonstrates the critical role of HIP2 in reducing cell death throughout nephrogenesis and maturation in wild-type mice and indicates a possible connection between decreased HIP2 expression in postnatal kidney structures and observed podocyte injury in yotari. Our results emphasize the crucial function of the examined markers throughout normal kidney development and their potential participation in kidney pathology and diagnostics, where they might serve as biomarkers and therapeutic targets.


Assuntos
Rim/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Biomarcadores/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Transdução de Sinais/fisiologia , Anormalidades Urogenitais/metabolismo , Refluxo Vesicoureteral/metabolismo
7.
Int J Mol Sci ; 23(11)2022 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-35682927

RESUMO

The aim of this study was to determine the effects of altered ganglioside composition on the expression of Cx37, Cx40, Cx43, Cx45, and Panx1 in different kidney regions of St8sia1 gene knockout mice (St8sia1 KO) lacking the GD3 synthase enzyme. Experiments were performed in twelve male 6-month-old mice: four wild-type (C57BL/6-type, WT) and eight St8sia1 KO mice. After euthanasia, kidney tissue was harvested, embedded in paraffin wax, and processed for immunohistochemistry. The expression of connexins and Panx1 was determined in different regions of the kidney: cortex (CTX.), outer stripe of outer medulla (O.S.), inner stripe of outer medulla (IN.S.), and inner medulla (IN.MED.). We determined significantly lower expression of Cx37, Cx40, Cx45, and Panx1 in different parts of the kidneys of St8sia1 KO mice compared with WT. The most consistent decrease was found in the O.S. where all markers (Cx 37, 40, 45 and Panx1) were disrupted in St8si1 KO mice. In the CTX. region, we observed decrease in the expression of Cx37, Cx45, and Panx1, while reduced expression of Cx37 and Panx1 was more specific to IN.S. The results of the present study suggest that deficiency of GD3 synthase in St8sia1 KO mice leads to disruption of renal Cx expression, which is probably related to alteration of ganglioside composition.


Assuntos
Conexinas , Rim , Animais , Conexinas/genética , Conexinas/metabolismo , Gangliosídeos/metabolismo , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Óxido Nítrico Sintase/metabolismo
8.
Int J Mol Sci ; 23(24)2022 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-36555181

RESUMO

This study aimed to explore the spatio-temporal expression patterns of congenital anomalies of kidney and urinary tract (CAKUT) candidate genes, Fibroblast Growth Factor Receptor 1 (FGFR1), Fibroblast Growth Factor Receptor 2 (FGFR2) and Receptor-Interacting Protein Kinase 5 (RIP5), in human fetal kidney development (CTRL) and kidneys affected with CAKUT. Human fetal kidneys from the 22nd to 41st developmental week (duplex, hypoplastic, dysplastic, and controls) were stained with antibodies and analyzed by epifluorescence microscopy and RT-qPCR. The effect of CAKUT candidate genes on kidney nephrogenesis and function is confirmed by statistically significant variations in the spatio-temporal expression patterns of the investigated markers. The nuclear localization of FGFR1, elevated expression score of FGFR1 mRNA, the increased area percentage of FGFR1-positive cells in the kidney cortex, and the overall decrease in the expression after the peak at the 27th developmental week in dysplastic kidneys (DYS), suggest an altered expression pattern and protein function in response to CAKUT pathophysiology. The RT-qPCR analysis revealed a significantly higher FGFR2 mRNA expression score in the CAKUT kidneys compared to the CTRL. This increase could be due to the repair mechanism involving the downstream mediator, Extracellular Signal-Regulated Kinase 1/2 (ERK1/2). The expression of RIP5 during normal human kidney development was reduced temporarily, due to urine production and increased later since it undertakes additional functions in the maturation of the postnatal kidney and homeostasis, while the expression dynamics in CAKUT-affected kidneys exhibited a decrease in the percentage of RIP5-positive cells during the investigated developmental period. Our findings highlight the importance of FGFR1, FGFR2, and RIP5 as markers in normal and pathological kidney development.


Assuntos
Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Proteína Serina-Treonina Quinases de Interação com Receptores , Sistema Urinário , Anormalidades Urogenitais , Humanos , Rim/fisiopatologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , RNA Mensageiro/genética , Sistema Urinário/anormalidades , Anormalidades Urogenitais/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética
9.
Int J Mol Sci ; 22(17)2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34502088

RESUMO

We aimed to investigate the spatio-temporal expression of possible CAKUT candidate genes CRKL, AIFM3, and UBASH3A, as well as AIF and BCL2 during human kidney development. Human fetal kidney tissue was stained with antibodies and analyzed by fluorescence microscopy and RT-PCR. Quantification of positive cells was assessed by calculation of area percentage and counting cells in nephron structures. Results showed statistically significant differences in the temporal expression patterns of the examined markers, depending on the investigated developmental stage. Limited but strong expression of CRKL was seen in developing kidneys, with increasing expression up to the period where the majority of nephrons are formed. Results also lead us to conclude that AIFM3 and AIF are important for promoting cell survival, but only AIFM3 is considered a CAKUT candidate gene due to the lack of AIF in nephron developmental structures. Our findings imply great importance of AIFM3 in energy production in nephrogenesis and tubular maturation. UBASH3A raw scores showed greater immunoreactivity in developing structures than mature ones which would point to a meaningful role in nephrogenesis. The fact that mRNA and proteins of CRKL, UBASH3A, and AIFM3 were detected in all phases of kidney development implies their role as renal development control genes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Rim/metabolismo , Proteínas Mitocondriais/genética , Anormalidades Urogenitais/genética , Refluxo Vesicoureteral/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fator de Indução de Apoptose/genética , Fator de Indução de Apoptose/metabolismo , Feto/embriologia , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Lactente , Recém-Nascido , Rim/embriologia , Rim/crescimento & desenvolvimento , Proteínas Mitocondriais/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
10.
Int J Mol Sci ; 22(7)2021 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-33800671

RESUMO

The spatiotemporal expression of α-tubulin, inversin and dishevelled-1 (DVL-1) proteins associated with the Wnt-signaling pathway, and primary cilia morphology were analyzed in developing kidneys (14th-38th developmental weeks), healthy postnatal (1.5- and 7-years old) and pathologically changed human kidneys, including multicystic dysplastic kidneys (MCDK), focal segmental glomerulosclerosis (FSGS) and nephrotic syndrome of the Finnish type (CNF). The analysis was performed by double immunofluorescence, electron microscopy, semiquantitative and statistical methods. Cytoplasmic co-expression of α-tubulin, inversin and DVL-1 was observed in the proximal convoluted tubules (pct), distal convoluted tubules (dct) and glomeruli (g) of analyzed tissues. During kidney development, the overall expression of α-tubulin, inversin and DVL-1 decreased, while in the postnatal period slightly increased. The highest expressions of α-tubulin and inversin characterized dct and g, while high DVL-1 characterized pct. α-tubulin, inversin and DVL-1 expression pattern in MCDK, FSGS and CNF kidneys significantly differed from the healthy control. Compared to healthy kidneys, pathologically changed kidneys had dysmorphic primary cilia. Different expression dynamics of α-tubulin, inversin and DVL-1 during kidney development could indicate that switch between the canonical and noncanonical Wnt-signaling is essential for normal kidney morphogenesis. In contrast, their disturbed expression in pathological kidneys might be associated with abnormal primary cilia, leading to chronic kidney diseases.


Assuntos
Cílios/metabolismo , Proteínas Desgrenhadas/metabolismo , Rim/embriologia , Rim/patologia , Fatores de Transcrição/metabolismo , Tubulina (Proteína)/metabolismo , Criança , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Glomerulosclerose Segmentar e Focal/metabolismo , Humanos , Lactente , Túbulos Renais/metabolismo , Rim Displásico Multicístico/metabolismo , Síndrome Nefrótica/metabolismo , Transdução de Sinais
11.
Int J Mol Sci ; 22(3)2021 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-33525532

RESUMO

Numerous evidence corroborates roles of gap junctions/hemichannels in proper kidney development. We analyzed how Dab1 gene functional silencing influences expression and localization of Cx37, Cx40, Cx43, Cx45, Panx1 and renin in postnatal kidneys of yotari mice, by using immunohistochemistry and electron microscopy. Dab1 Δ102/221 might lead to the activation of c-Src tyrosine kinase, causing the upregulation of Cx43 in the medulla of yotari mice. The expression of renin was more prominent in yotari mice (p < 0.001). Renin granules were unusually present inside the vascular walls of glomeruli capillaries, in proximal and distal convoluted tubules and in the medulla. Disfunction of Cx40 is likely responsible for increased atypically positioned renin cells which release renin in an uncontrolled fashion, but this doesn't rule out simultaneous involvement of other Cxs, such as Cx45 which was significantly increased in the yotari cortex. The decreased Cx37 expression in yotari medulla might contribute to hypertension reduction provoked by high renin expression. These findings imply the relevance of Cxs/Panx1 as markers of impaired kidney function (high renin) in yotari mice and that they have a role in the preservation of intercellular signaling and implicate connexopathies as the cause of premature death of yotari mice.


Assuntos
Conexina 43/genética , Conexinas/genética , Glomérulos Renais/metabolismo , Proteínas do Tecido Nervoso/genética , Renina/genética , Animais , Animais Recém-Nascidos , Conexina 43/metabolismo , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Junções Comunicantes/patologia , Regulação da Expressão Gênica no Desenvolvimento , Glomérulos Renais/crescimento & desenvolvimento , Glomérulos Renais/patologia , Medula Renal/crescimento & desenvolvimento , Medula Renal/metabolismo , Medula Renal/patologia , Túbulos Renais/crescimento & desenvolvimento , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/metabolismo , Renina/metabolismo , Transdução de Sinais , Proteína alfa-5 de Junções Comunicantes , Proteína alfa-4 de Junções Comunicantes
12.
Cell Tissue Res ; 378(2): 301-317, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31256287

RESUMO

The aim was to explore the influence of experimental diabetes mellitus type 1 (DM1) and potential protective/deleterious effects of different dietary n-6/n-3 PUFA ratios on renal phospholipid composition and pathological changes caused by DM1. Male Wistar rats were injected with 55 mg/kg streptozotocin or citrate buffer (control group). Control (C) and diabetic groups (STZ) were fed with n-6/n-3 ratio of ≈ 7, STZ + N6 with n-6/n-3 ratio ≈ 60 and STZ + DHA with n-6/n-3 ratio of ≈ 1 containing 16% EPA and 19% DHA. Tissues were harvested 30 days after DM1 induction. Blood and kidneys were collected and analysed for phospholipid fatty acid composition, pathohystological changes, ectopic lipid accumulation and expression of VEGF, NF-kB and special AT-rich sequence-binding protein-1 (SATB1). Pathological changes were studied also by using transmission electron microscopy, after immunostaining for VEGF. Substantial changes in renal phospholipid fatty acid composition resulted from DM1 and dietary PUFA manipulation. Extensive vacuolization of distal tubular cells (DTCs) was found in DM1, but it was attenuated in the STZ + DHA group, in which the highest renal NF-kB expression was observed. The ectopic lipid accumulation was observed in proximal tubular cells (PTCs) of all diabetic animals, but it was worsened in the STZ + N6 group. In DM1, we found disturbance of VEGF-transporting vesicular PTCs system, which was substantially worsened in STZ + DHA and STZ + N6. Results have shown that the early phase of DN is characterized with extent damage and vacuolization of DTCs, which could be attenuated by DHA/EPA supplementation. We concluded that dietary fatty acid composition can strongly influence the outcomes of DN.


Assuntos
Diabetes Mellitus Tipo 1/patologia , Nefropatias Diabéticas/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Túbulos Renais Distais , Animais , Diabetes Mellitus Experimental , Suplementos Nutricionais , Ácidos Graxos/metabolismo , Proteínas de Homeodomínio/metabolismo , Túbulos Renais Distais/metabolismo , Túbulos Renais Distais/patologia , Masculino , Fosfolipídeos/metabolismo , Ratos , Ratos Wistar , Fator A de Crescimento do Endotélio Vascular/metabolismo
13.
Croat Med J ; 60(6): 521-531, 2019 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-31894918

RESUMO

AIM: To explore the spatial and temporal expression patterns of DAB1 and Reelin in the developing and postnatal healthy human kidneys as potential determinants of kidney development. METHODS: Paraffin-embedded fetal kidney tissue between the 13/14th and 38th developmental weeks (dw) and postnatal tissue at 1.5 and 7 years were stained with DAB1 and Reelin antibodies by double immunofluorescence. RESULTS: During the fetal kidney development and postnatal period, DAB1 and Reelin showed specific spatial expression pattern and diverse fluorescence intensity. During the fetal period, DAB1 was strongly expressed in the distal convoluted tubules (DCT), with strong reactivity, and diversely in the proximal convoluted tubules (PCT) and glomeruli. In the postnatal period, DAB1 expression decreased. The strongest Reelin expression in early fetal stages was observed in the PCT. In the postnatal period, Reelin expression decreased dramatically in all observed structures. These two markers were colocalized during early developmental stages, mostly in PCT, DCT, and podocytes. CONCLUSION: The appearance of DAB1 and Reelin during fetal kidney development confirms their potential significant role in the formation of kidney structure or function. High DAB1 expression in the DCT implies its regulatory role in tubular formation or function maintenance during development. Reelin was highly expressed in human kidneys at early fetal stages, mostly in the PCT, while at later fetal stages and postnatal period its expression decreased.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Rim/embriologia , Rim/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/metabolismo , Criança , Desenvolvimento Fetal , Idade Gestacional , Humanos , Lactente , Rim/metabolismo , Túbulos Renais Distais/embriologia , Túbulos Renais Distais/metabolismo , Túbulos Renais Proximais/embriologia , Túbulos Renais Proximais/metabolismo , Podócitos/metabolismo , Proteína Reelina
14.
Biomedicines ; 12(7)2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-39062115

RESUMO

Our study examines the immunoexpression patterns of Megalin, Cubilin, Caveolin-1, Gipc1 and Dab2IP in the embryonic development (E) and postnatal (P) mouse kidney, with a focus on differentiating patterns between wild-type (wt) and yotari, Dab1-/- (yot) mice. Immunofluorescence revealed raised immunoexpression of receptors Megalin and Cubilin at the ampulla/collecting ducts and convoluted tubules across all developmental stages, with the most prominent immunoexpression observed in the convoluted tubules and the parietal epithelium of the Bowman's capsule. Quantitative analysis showed a higher percentage of Megalin and Cubilin in wt compared to yot mice at E13.5. Co-expression of Megalin and Cubilin was observed at the apical membrane of convoluted tubules and the parietal layer of the Bowman's capsule. The staining intensity of Megalin varied across developmental stages, with the strongest reactivity observed at the ampulla and collecting ducts at embryonic day (E) 13.5 in wt mice. In contrast, Caveolin-1 exhibited high immunoexpression in the metanephric mesenchyme, blood vessels, and the border area between the metanephric mesenchyme and renal vesicle, with a decrease in immunoexpression as development progressed. Gipc1 showed diffuse cytoplasmic staining in metanephric mesenchyme, convoluted tubules and collecting ducts, with significant differences in immunoexpression between wild-type and yot mice at both investigated embryonic time points. Dab2IP immunofluorescent staining was most prominent in renal vesicle/glomeruli and ampulla/collecting ducts at E13.5, with mild staining intensity observed in the distal convoluted tubules postnatally. Our findings elucidate distinct immunoexpression of patterns and potential parts of these proteins in the development and function of the kidney, highlighting the importance of further investigation into their regulatory mechanisms.

15.
Life (Basel) ; 14(3)2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38541642

RESUMO

The purpose of this study was to evaluate the effects of Dab1 gene silencing on the immunoexpression of light chain 3 beta (Lc3b), glucose regulating protein 78 (Grp78), heat shock cognate 71 (Hsc70), mammalian target of rapamycin (mTOR) and lysosomal-associated membrane protein 2A (Lamp2a) in the lung tissue of developing yotari (Dab1-/-) and wild-type (wt) mice. The lung epithelium and mesenchyme of the embryos at gestational days E13.5 and E15.5 were examined using immunofluorescence and semi-quantitative methods. In the pulmonary mesenchyme and epithelium, Grp78 and Lc3b of moderate fluorescence reactivity was demonstrated in wt mice for both evaluated time points, while yotari mice exhibited only epithelial reactivity for the same markers. Mild punctate expression of Hsc70 was observed for both genotypes. A significant difference was present when analyzing mTOR expression, where wt mice showed strong perinuclear staining in the epithelium. According to our data, Dab1 gene silencing may result in autophagy abnormalities, which could then cause respiratory system pathologies via defective lung cell degradation by lysosome-dependent cell elimination.

16.
Cells ; 13(17)2024 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-39273022

RESUMO

Melanoma is the most severe type of skin cancer and among the most malignant neoplasms in humans. With the growing incidence of melanoma, increased numbers of therapeutic options, and the potential to target specific proteins, understanding the basic mechanisms underlying the disease's progression and resistance to treatment has never been more important. LOXL3, SNAI1, and NES are key factors in melanoma genesis, regulating tumor growth, metastasis, and cellular differentiation. In our study, we explored the potential role of LOXL3, SNAI1, and NES in melanoma progression and metastasis among patients with dysplastic nevi, melanoma in situ, and BRAF+ and BRAF- metastatic melanoma, using immunofluorescence and qPCR analysis. Our results reveal a significant increase in LOXL3 expression and the highest NES expression in BRAF+ melanoma compared to BRAF-, dysplastic nevi, and melanoma in situ. As for SNAI1, the highest expression was observed in the metastatic melanoma group, without significant differences among groups. We found co-expression of LOXL3 and SNAI1 in the perinuclear area of all investigated subgroups and NES and SNAI1 co-expression in melanoma cells. These findings suggest a codependence or collaboration between these markers in melanoma EMT, suggesting new potential therapeutic interventions to block the EMT cascade that could significantly affect survival in many melanoma patients.


Assuntos
Progressão da Doença , Melanoma , Fatores de Transcrição da Família Snail , Melanoma/genética , Melanoma/patologia , Melanoma/metabolismo , Humanos , Fatores de Transcrição da Família Snail/metabolismo , Fatores de Transcrição da Família Snail/genética , Regulação Neoplásica da Expressão Gênica , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/metabolismo , Linhagem Celular Tumoral , Masculino , Feminino , Metástase Neoplásica , Pessoa de Meia-Idade
17.
Biomolecules ; 14(9)2024 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-39334940

RESUMO

This study aimed to explore how Dab1 functional silencing influences the expression patterns of different connexins in the developing yotari (yot) mice eyes as potential determinants of retinogenesis. Using immunofluorescence staining, the protein expression of Dab1, Reelin, and connexin 37, 40, 43, and 45 (Cx37, Cx40, Cx43, and Cx45) in the wild-type (wt) and yot eyes at embryonic days 13.5 and 15.5 (E13.5 and E15.5) were analyzed. Different expression patterns of Cx37 were seen between the wt and yot groups. The highest fluorescence intensity of Cx37 was observed in the yot animals at E15.5. Cx40 had higher expression at the E13.5 when differentiation of retinal layers was still beginning, whereas it decreased at the E15.5 when differentiation was at the advanced stage. Higher expression of Cx43 was found in the yot group at both time points. Cx45 was predominantly expressed at E13.5 in both groups. Our results reveal the altered expression of connexins during retinogenesis in yot mice and their potential involvement in retinal pathology, where they might serve as prospective therapeutic targets.


Assuntos
Conexinas , Proteína Reelina , Animais , Camundongos , Conexinas/metabolismo , Conexinas/genética , Proteína Reelina/metabolismo , Retina/metabolismo , Retina/crescimento & desenvolvimento , Olho/metabolismo , Olho/embriologia , Olho/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Diferenciação Celular
18.
Genes (Basel) ; 15(6)2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38927638

RESUMO

Approximately half of the cases of chronic kidney disease (CKD) in childhood are caused by congenital anomalies of the kidney and urinary tract (CAKUT). Specific genes were identified as having significant importance in regard to the underlying genetic factors responsible for the CAKUT phenotype, and in our research, we focused on analyzing and comparing the expression levels of ectodysplasin A2 receptor (EDA2R), protocadherin9 (PCDH9), and TNF receptor-associated factor 7 (TRAF7) proteins in the cortex and medulla of healthy control kidneys during developmental phases 2, 3, and 4. We also performed an analysis of the area percentages of the mentioned proteins in the cortical and medullary sections of healthy embryonic and fetal kidneys compared to those affected by CAKUT, including duplex kidneys (DK), horseshoe kidneys (HK), hypoplastic kidneys (HYP), and dysplastic kidneys (DYS). We found that the CAKUT candidate gene proteins EDA2R, PCDH9, and TRAF7 are all expressed during normal human kidney development stages. In DYS, the expression of EDA2R was higher than in normal kidneys, likely due to EDA2R's role in apoptosis, which was upregulated in specific cases and could possibly contribute to the formation of DYS. The expression of PCDH9 was lower in HK, which can be attributed to the possible role of PCDH9 in cell migration suppression. Decreased PCDH9 expression is linked to increased cell migration, potentially contributing to the development of HK. The level of TRAF7 expression was reduced in all examined kidney disorders compared to normal kidneys, suggesting that this reduction might be attributed to the crucial role of TRAF7 in the formation of endothelium and ciliogenesis, both of which are essential for normal kidney development. Further research is required to ascertain the function of these proteins in both the typical development of the kidney and in CAKUT.


Assuntos
Caderinas , Rim , Anormalidades Urogenitais , Refluxo Vesicoureteral , Humanos , Caderinas/genética , Caderinas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Rim/metabolismo , Rim/anormalidades , Rim/crescimento & desenvolvimento , Rim/embriologia , Protocaderinas , Sistema Urinário/anormalidades , Sistema Urinário/metabolismo , Anormalidades Urogenitais/genética , Anormalidades Urogenitais/patologia , Refluxo Vesicoureteral/genética , Refluxo Vesicoureteral/patologia
19.
Biomolecules ; 13(3)2023 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-36979337

RESUMO

The purpose of this study was to compare the immunofluorescence patterns of autophagic markers: Light chain 3 beta (LC3B), Glucose regulating protein 78 (GRP78), Heat shock cognate 71 (HSC70) and Lysosomal-associated membrane protein 2A (LAMP2A) in the developing and postnatal kidneys of Dab1-/- (yotari) mice to those of wild-type samples. Embryos were obtained on gestation days 13.5 and 15.5 (E13.5 and E15.5), and adult animals were sacrificed at postnatal days 4, 11 and 14 (P4, P11, and P14). After fixation and dehydration, paraffin-embedded kidney tissues were sectioned and incubated with specific antibodies. Using an immunofluorescence microscope, sections were analyzed. For statistical analysis, a two-way ANOVA test and a Tukey's multiple comparison test were performed with a probability level of p < 0.05. A significant increase in GRP78 and LAMP2A expression was observed in the renal vesicles and convoluted tubules of yotari in embryonic stages. In postnatal kidneys, all observed proteins showed higher signal intensities in proximal and distal convoluted tubules of yotari, while a higher percentage of LC3B-positive cells was also observed in glomeruli. Our findings suggest that all of the examined autophagic markers play an important role in normal kidney development, as well as the potential importance of these proteins in renal pathology, where they primarily serve a protective function and thus may be used as diagnostic and therapeutic targets.


Assuntos
Chaperona BiP do Retículo Endoplasmático , Rim , Animais , Camundongos , Rim/metabolismo , Túbulos Renais , Autofagia , Proteínas do Tecido Nervoso/metabolismo
20.
Genes (Basel) ; 14(2)2023 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-36833374

RESUMO

Approximately 60% of patients with squamous cell carcinoma (LSCC) have regional occult metastatic disease/distant metastases at the time of diagnosis, putting them at higher risk for disease progression. Therefore, biomarkers are needed for early prognostic purpose. The aim of this study was to analyze the expression pattern of connexins (Cx) 37, 40 and 45, pannexin1 (Panx1) and vimentin in LSCC and correlate with tumor grade (G) and outcome. METHODS: Thirty-four patients who underwent (hemi-)laryngectomy and regional lymphadenectomy due to LSCC from 2017 to 2018 in University Hospital Split, Croatia, were studied. Samples of tumor tissue and adjacent normal mucosa embedded in paraffin blocks were stained using the immunofluorescence method and were semi-quantitatively analyzed. RESULTS: The expression of Cx37, Cx40, and Panx1 differed between cancer and adjacent normal mucosa and between histological grades, being the highest in well-differentiated (G1) cancer and low/absent in poorly differentiated (G3) cancer (all p < 0.05). The expression of vimentin was the highest in G3 cancer. Expression of Cx45 was generally weak/absent, with no significant difference between cancer and the controls or between grades. Lower Panx1 and higher vimentin expression were found to be prognostic factors for regional metastatic disease. Lower Cx37 and 40 expressions were present in patients with disease recurrence after the three-year follow-up period. CONCLUSION: Cx37 and Cx40, Panx1, and vimentin have the potential to be used as prognostic biomarkers for LSCC.


Assuntos
Neoplasias de Cabeça e Pescoço , Recidiva Local de Neoplasia , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço , Vimentina , Conexinas/metabolismo , Proteínas do Tecido Nervoso
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