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1.
Biochemistry (Mosc) ; 84(12): 1502-1512, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31870254

RESUMO

Autophagy plays an important role in the pathogenesis of acute kidney injury (AKI). Although autophagy activation was shown to be associated with an increased lifespan and beneficial effects in various pathologies, the impact of autophagy activators, particularly, rapamycin and its analogues on AKI remains obscure. In our study, we explored the effects of rapamycin treatment in in vivo and in vitro models of ischemic and cisplatin-induced AKI. The impact of rapamycin on the kidney function after renal ischemia/reperfusion (I/R) or exposure to the nephrotoxic agent cisplatin was assessed by quantifying blood urea nitrogen and serum creatinine and evaluating the content of neutrophil gelatinase-associated lipocalin, a novel biomarker of AKI. In vitro experiments were performed on the primary culture of renal tubular cells (RTCs) that were subjected to oxygen-glucose deprivation (OGD) or incubated with cisplatin under various rapamycin treatment protocols. Cell viability and proliferation were estimated by the MTT assay and real-time cell analysis using an RTCA iCELLigence system. Although rapamycin inhibited mTOR (mammalian target of rapamycin) signaling, it failed to enhance the autophagy and to ameliorate the severity of AKI caused by ischemia or cisplatin-induced nephrotoxicity. Experiments with RTCs demonstrated that rapamycin exhibited the anti-proliferative effect in primary RTCs cultures but did not protect renal cells exposed to OGD or cisplatin. Our study revealed for the first time that the mTOR inhibitor rapamycin did not prevent AKI caused by renal I/R or cisplatin-induced nephrotoxicity and, therefore, cannot be considered as an ideal mimetic of the autophagy-associated nephroprotective mechanisms (e.g., those induced by caloric restriction), as it had been suggested earlier. The protective action of such approaches like caloric restriction might not be limited to mTOR inhibition and can proceed through more complex mechanisms involving alternative autophagy-related targets. Thus, the use of rapamycin and its analogues for the treatment of various AKI forms requires further studies in order to understand potential protective or adverse effects of these compounds in different contexts.


Assuntos
Lesão Renal Aguda/induzido quimicamente , Lesão Renal Aguda/prevenção & controle , Antineoplásicos/efeitos adversos , Cisplatino/efeitos adversos , Isquemia/prevenção & controle , Sirolimo/farmacologia , Lesão Renal Aguda/metabolismo , Animais , Células Cultivadas , Glucose/metabolismo , Isquemia/metabolismo , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Masculino , Oxigênio/metabolismo , Substâncias Protetoras/farmacologia , Ratos , Serina-Treonina Quinases TOR/metabolismo
2.
Life Sci ; 238: 116957, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31655195

RESUMO

Epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) deposition in renal tubular epithelial cells are critical to diabetic nephropathy (DN) pathogenesis, but the underlying mechanisms remain undefined. Bone morphogenetic protein 7 (BMP-7) inhibits EMT and ECM accumulation in renal tubular epithelial cells cultured in presence of high glucose. Meanwhile, miRNA-21 (miR-21) downregulates Smad7, promoting EMT and ECM deposition. However, the association of BMP-7 with miR-21/Smad7 in DN is unknown. Here, NRK-52E cells incubated in presence of high glucose and STZ-induced C57BL diabetic mice were considered in vitro and in vivo models of DN, respectively. In both models, BMP-7 (mRNA/protein) amounts were decreased as well as Smad7 protein expression, while miR-21 expression and TGF-ß1/Smad3 pathway activation were enhanced, accompanied by enhanced EMT and ECM deposition. Further, addition of BMP-7 human recombinant cytokine (rhBMP-7) and injection of the BMP-7 overexpression plasmid in diabetic mice markedly downregulated miR-21 and upregulated Smad7, reduced Smad3 activation without affecting TGF-ß1 amounts, and prevented EMT and ECM accumulation. MiR-21 overexpression in the in vitro model downregulated Smad7, promoted EMT and ECM accumulation without affecting BMP-7 amounts, and miR-21 downregulation reversed it. By interfering with BMP-7 and miR-21 expression in high glucose conditions, miR-21 amounts and Smad3 phosphorylation were further decreased. Smad7 was then upregulated, and EMT and ECM deposition were inhibited; these effects were reversed after miR-21 overexpression. These findings suggest that BMP-7 decreases renal fibrosis in DN by regulating miR-21/Smad7 signaling, providing a theoretical basis for the development of novel and effective therapeutic drugs for DN.


Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Nefropatias Diabéticas/complicações , Fibrose/prevenção & controle , Regulação da Expressão Gênica , Túbulos Renais/metabolismo , MicroRNAs/antagonistas & inibidores , Animais , Proteína Morfogenética Óssea 7/genética , Células Cultivadas , Transição Epitelial-Mesenquimal , Matriz Extracelular/metabolismo , Fibrose/etiologia , Fibrose/patologia , Túbulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Fosforilação , Transdução de Sinais , Proteína Smad7/genética , Proteína Smad7/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
3.
Food Chem Toxicol ; 133: 110720, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31369848

RESUMO

The incidence of kidney disease has increased rapidly in recent years. One major possible reason for this increase in nephrosis is from foodborne toxins. Since the mechanism of how foodborne toxins are involved in the process of nephrosis is largely unknown, the current study aims to establish a profile for how one of the major toxin threats, ochratoxin A (OTA), induce differential protein expression. In this proteomic study of rat kidneys, 75 kd glucose-regulated protein (Grp75) expression was found to be sensitized by a low concentration of OTA, but inhibited by high doses. In response to OTA, a decrease in Grp75 expression preceded the inhibition of mitochondrial Lon peptidase 1 (Lonp1). Using Grp75 knockdown cell line, it was shown that the inhibition of Grp75 promoted the secretion of kidney injury molecule 1 (Kim1), and suppressed Lonp1 expression in renal injury. Moreover, the acceleration of renal disease was associated with the consumption of Grp75. Our study suggests that the Grp75 protein may be valuable as both a treatment and biomarker for the foodborne diseases that induce renal tubular necrosis. The findings of this research are beneficial for the establishment of nutritional interventions, and the screening of therapeutic targets, in cases of nephrosis.


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Túbulos Renais/metabolismo , Proteínas Mitocondriais/metabolismo , Proteases Dependentes de ATP/metabolismo , Animais , Biomarcadores/metabolismo , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Doenças Transmitidas por Alimentos/metabolismo , Doenças Transmitidas por Alimentos/patologia , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP70/genética , Humanos , Túbulos Renais/patologia , Masculino , Proteínas Mitocondriais/genética , Necrose , Ocratoxinas , Proteômica/métodos , Ratos Endogâmicos F344 , Ratos Wistar , Transcriptoma
4.
J Diabetes Res ; 2019: 2510105, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31467925

RESUMO

Background: Epithelial-to-mesenchymal transition (EMT) is thought to play a significant role in the advancement to chronic kidney disease and contributes to the deposition of extracellular matrix proteins and renal fibrosis relating to diabetic nephropathy. Method: We studied the effect of Nrf2-HO-1 signaling on high-glucose- (HG-) induced EMT in normal human tubular epithelial cells, that is, HK2 cells. In short, we treated HK2 cells with HG and sulforaphane (SFN) as an Nrf2 activator. EMT was evaluated by the expression activity of the epithelial marker E-cadherin and mesenchymal markers such as vimentin and fibronectin. Results: Exposure of HK2 cells to HG (60 mM) activated the expression of vimentin and fibronectin but decreased E-cadherin. Treatment of HK2 cells with SFN caused HG-induced attenuation in EMT markers with activated Nrf2-HO-1. We found that SFN decreased HG-induced production of reactive oxygen species (ROS), phosphorylation of PI3K/Akt at serine 473, and inhibitory phosphorylation of serine/threonine kinase glycogen synthase kinase-3ß (GSK-3ß) at serine 9. Subsequently, these signaling led to the downregulation of the Snail-1 transcriptional factor and the recovery of E-cadherin. Conclusion: The present study suggests that Nrf2-HO-1 signaling has an inhibitory role in the regulation of EMT through the modulation of ROS-mediated PI3K/Akt/GSK-3ß activity, highlighting Nrf2-HO-1 and GSK-3ß as potential therapeutic targets in diabetic nephropathy.


Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glucose/farmacologia , Heme Oxigenase-1/fisiologia , Túbulos Renais/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/fisiologia , Espécies Reativas de Oxigênio/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Transição Epitelial-Mesenquimal/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HEK293 , Humanos , Túbulos Renais/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
5.
Curr Top Med Chem ; 19(22): 2058-2068, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31400266

RESUMO

BACKGROUND: Envenomation caused by Bothrops alternatus is common in Southern Brazil. Acute Kidney Injury occurs after Bothrops snakebite and more information is necessaryrequired to understand its mechanism. OBJECTIVE: The objective was to evaluate the effect of Bothrops alternatus venom (BaV) on renal cells and rat isolated kidney function. METHODS: Wistar rats (n = 6, weighing 260-320 g) were perfused with a Krebs-Henseleit solution containing 6 g 100 mL-1 of bovine serum albumin. After 30 minutes, the kidneys were perfused with BaV to a final concentration of 1 and 3 µgmL-1; and subsequently were evaluated for Perfusion Pressure (PP), Renal Vascular Resistance (RVR), Urinary Flow (UF), Glomerular Filtration Rate (GFR), and percentage of electrolyte tubular transport. Renal histological analysis, cytokine release, oxidative stress and cytotoxicity in renal proximal tubular cells were assessed. RESULTS: BaV reduced PP, RVR, GFR, UF, total and proximal sodium transport (%TNa+), and chloride (%TCl-) in the isolated kidney perfusion model. Histological analysis of perfused kidneys disclosed the presence of proteinaceous material in the glomeruli and renal tubules, vacuolar tubular epithelial cell degeneration, Bowman's capsule degeneration, swelling of glomerular epithelial cells, glomerular atrophy and degeneration, and the presence of intratubular protein. Cytokine release (TNF-α, IL-1ß, IL-10) and oxidative stress were increased in the kidneys. The viability of LLC-MK2 cells (IC50: 221.3 µg/mL) was decreased by BaV and necrosis was involved in cell death. CONCLUSION: These findings indicate that BaV modifies functional parameters in an isolated perfused kidney model and has cytotoxic effects on renal lineage cells.


Assuntos
Citocinas/biossíntese , Túbulos Renais/efeitos dos fármacos , Venenos de Serpentes/farmacologia , Animais , Bothrops , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Taxa de Filtração Glomerular , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Macaca mulatta , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Relação Estrutura-Atividade
6.
Int J Mol Sci ; 20(15)2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31390839

RESUMO

Despite the wide use of angiotensin II receptor blockers in the treatment of Alport syndrome (AS), the mechanism as to how angiotensin II receptor blockers prevent interstitial fibrosis remains unclear. Here, we report that treatment of olmesartan effectively targets the feedback loop between the renin-angiotensin system (RAS) and transforming growth factor ß (TGFß) signals in tubular epithelial cells and preserves renal angiotensin-converting enzyme 2 (ACE2) expression in the kidney of Col4a3-/- mice, a murine model of experimental AS. Morphology analyses revealed amelioration of kidney fibrosis in Col4a3-/- mice by olmesartan treatment. Upregulation of TGFß and activation of its downstream in Col4a3-/- mice were attenuated by olmesartan in Col4a3-/- mice. Intriguingly, TGFß expression was preferentially upregulated in damaged tubular epithelial cells in Col4a3-/- mice. Concurrent upregulation of TNFα-converting enzyme and downregulation of ACE2 suggested RAS activation in Col4a3-/- mice, which was prevented by olmesartan. Mechanistically, olmesartan suppressed TGFß-induced RAS activation in tubular epithelial cells in vitro. Collectively, we concluded that olmesartan effectively suppresses the progression of tubulointerstitial fibrosis in AS by interrupting RAS-TGFß feedback loop to counterbalance intrarenal RAS activation.


Assuntos
Anti-Hipertensivos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imidazóis/farmacologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Nefrite Hereditária/genética , Nefrite Hereditária/patologia , Tetrazóis/farmacologia , Fator de Crescimento Transformador beta/genética , Animais , Apoptose/efeitos dos fármacos , Biomarcadores , Biópsia , Modelos Animais de Doenças , Fibrose , Túbulos Renais/patologia , Camundongos , Camundongos Knockout , Nefrite Hereditária/tratamento farmacológico , Nefrite Hereditária/metabolismo , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Resultado do Tratamento , Proteínas ras/genética , Proteínas ras/metabolismo
7.
Mol Med Rep ; 20(2): 1613-1620, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31257491

RESUMO

Renal tubular epithelial cell apoptosis is an important pathological mechanism of septic acute kidney injury (AKI). Endotoxin, also known as lipopolysaccharide (LPS), has a key role in septic AKI and can directly induce tubular epithelial cell apoptosis. The upregulation of receptor­interacting protein kinase 3 (RIPK3) in tubular epithelial cells has been reported in septic AKI, with RIPK3 mediating apoptosis in several cell types. In the present study, the effect of RIPK3 on endotoxin­induced AKI was investigated in mouse tubular epithelial cell apoptosis in vitro and in vivo. It was found that the expression of RIPK3 was markedly increased in endotoxin­induced AKI. Endotoxin­induced AKI and tubular epithelial cell apoptosis could be attenuated by GSK'872, a RIPK3 inhibitor. LPS stimulation also upregulated RIPK3 expression in tubular epithelial cells in a time­dependent manner. Both RIPK3 inhibitor and small interfering RNA (siRNA) targeting RIPK3 reduced LPS­induced tubular epithelial cell apoptosis in vitro. The expression of the proapoptotic protein Bax was induced by LPS and reversed by GSK'872 or RIPK3­siRNA. The present study revealed that RIPK3 mediated renal tubular cell apoptosis in endotoxin­induced AKI. RIPK3 may be a potential target for the prevention of renal tubular cell apoptosis in endotoxin­induced AKI.


Assuntos
Lesão Renal Aguda/metabolismo , Células Epiteliais/patologia , Túbulos Renais/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Lesão Renal Aguda/induzido quimicamente , Lesão Renal Aguda/patologia , Animais , Apoptose , Células Cultivadas , Modelos Animais de Doenças , Endotoxinas/efeitos adversos , Células Epiteliais/metabolismo , Túbulos Renais/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteína Serina-Treonina Quinases de Interação com Receptores/análise
8.
Int J Mol Sci ; 20(15)2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31357488

RESUMO

The use of donors deceased after brain death (DBD) with extended criteria in response to the shortage of grafts leads to the removal of more fragile kidneys. These grafts are at greater risk of not being grafted or delayed function. A better knowledge of the pathophysiology of DBDs would improve this situation. There is a difference between the results from animal models of DBD and the clinical data potentially explained by the kinetics of brain death induction. We compared the effect of the induction rate of brain death on the recovery of post-transplant renal function in a pig model of DBD followed by allografts in nephrectomized pigs. Resumption of early function post-transplant was better in the rapidly generated brain death group (RgBD) and graft fibrosis at three months less important. Two groups had identical oxidative stress intensity but a greater response to this oxidative stress by SIRT1, PGC1-α and NRF2 in the RgBD group. Modulation of mechanistic target of rapamycin (mTOR) stimulation by NRF2 would also regulate the survival/apoptosis balance of renal cells. For the first time we have shown that an allostatic response to oxidative stress can explain the impact of the rapidity of brain death induction on the quality of kidney transplants.


Assuntos
Morte Encefálica/metabolismo , Transplante de Rim , Rim/metabolismo , Animais , Biomarcadores , Função Retardada do Enxerto , Endotélio Vascular/metabolismo , Fibrose , Rim/patologia , Túbulos Renais/metabolismo , Modelos Animais , Estresse Oxidativo , Insuficiência Renal/etiologia , Insuficiência Renal/metabolismo , Insuficiência Renal/patologia , Suínos , Fatores de Tempo , Doadores de Tecidos , Transplante Homólogo
9.
Food Funct ; 10(6): 3782-3797, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31180394

RESUMO

Quercetin is the most ubiquitous flavonoid in fruits, herbs, vegetables and products made from them. It shows the potential to inhibit the progression of kidney fibrosis and the epithelial to mesenchymal transition (EMT) of the renal tubular system, but the molecular mechanism behind this is still not known. In our study, we explored the effect of quercetin treatment on extracellular matrix (ECM) deposition and stimulation of the EMT in vitro and in vivo and tried to deduce the mechanisms regulating these effects. In rats having unilateral ureter obstruction (UUO), quercetin treatment significantly prevented renal function decline. Quercetin reduced the TGF-ß1 expression and inhibited the epithelial cell to mesenchymal cell phenotypic switch, as well as ECM deposition in rats with UUO. In cultured epithelial cells of the renal tubular region (NRK-52E), quercetin markedly ameliorated the EMT and ECM synthesis induced by TGF-ß1. Activation of the Hedgehog pathway was closely related to EMT induction. Quercetin effectively suppressed the hyperactive Hedgehog pathway in NRK-52E cells treated with TGF-ß1 and in kidney obstructed rats, which reduced the EMT, ECM deposition and cellular proliferation. Moreover, we examined certain transcriptional factors (slug, snail, ZEB-1 and twist) that govern the E-cadherin expression at the level of transcription. The results unveiled that the four transcriptional factors were highly repressed in NRK-52E cells treated with TGF-ß1 and also in obstructed kidneys by quercetin-mediated inhibition. Therefore, these outcomes indicate that quercetin could alleviate fibrosis and the EMT in vitro and in vivo by inhibiting the activation of Hedgehog signaling and could act as a therapeutic agent for patients having several kinds of renal fibrotic diseases.


Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Nefropatias/tratamento farmacológico , Túbulos Renais/metabolismo , Quercetina/administração & dosagem , Animais , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibrose , Proteínas Hedgehog/genética , Humanos , Nefropatias/metabolismo , Nefropatias/patologia , Nefropatias/fisiopatologia , Túbulos Renais/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
10.
J Recept Signal Transduct Res ; 39(1): 73-79, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31184240

RESUMO

Terfenadine, an antihistamine used for the treatment of allergic conditions, affected Ca2+-related physiological responses in various models. However, the effect of terfenadine on cytosolic free Ca2+ levels ([Ca2+]i) and its related physiology in renal tubular cells is unknown. This study examined whether terfenadine altered Ca2+ signaling and caused cytotoxicity in Madin-Darby canine kidney (MDCK) renal tubular cells. The Ca2+-sensitive fluorescent dye fura-2 was used to measure [Ca2+]i. Cell viability was measured by the fluorescent reagent 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] water soluble tetrazolium-1 (WST-1) assay. Terfenadine at concentrations of 100-1000 µM induced [Ca2+]i rises concentration dependently. The response was reduced by approximately 35% by removing extracellular Ca2+. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) partly inhibited terfenadine-evoked [Ca2+]i rises. Conversely, treatment with terfenadine abolished BHQ-evoked [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 inhibited 95% of terfenadine-induced Ca2+ release. Terfenadine-induced Ca2+ entry was supported by Mn2+-caused quenching of fura-2 fluorescence. Terfenadine-induced Ca2+ entry was partly inhibited by an activator of protein kinase C (PKC), phorbol 12-myristate 13 acetate (PMA) and by three modulators of store-operated Ca2+ channels (nifedipine, econazole, and SKF96365). Terfenadine at 200-300 µM decreased cell viability, which was not reversed by pretreatment with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Together, in MDCK cells, terfenadine induced [Ca2+]i rises by evoking PLC-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via PKC-sensitive store-operated Ca2+ entry. Furthermore, terfenadine caused cell death that was not triggered by preceding [Ca2+]i rises.


Assuntos
Apoptose/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Antagonistas dos Receptores Histamínicos H1 não Sedativos/farmacologia , Túbulos Renais/patologia , Terfenadina/farmacologia , Animais , Sobrevivência Celular , Cães , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Células Madin Darby de Rim Canino
11.
Biomed Res Int ; 2019: 2821731, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31223614

RESUMO

Objective: Sepsis and associated acute kidney injury (SAKI) are determined to be closely related to poor prognosis. Because the metabolic alterations of tubular epithelial cells (TECs) are crucial for the occurrence and development of SAKI, we carried out this present study to identify the metabolism changes of TECs during SAKI and relevant mechanisms. Methods: Rat SAKI model and rat tubular epithelial cell line were used in our study. ELISA was used to determine the serum cytokines levels. Protein expressions were examined with Western-Blotting and the transcriptions of RNAs were determined with qRT-PCR. ADP/ATP assay and Oil Red O staining were used to examine the energy and lipid metabolism, respectively. Dual-luciferase reporter assay was carried out to determine the interactions between miRNA and specific proteins. Cell cycle arrest and apoptosis were determined with flow cytometry. Results: Sepsis and AKI were induced 12 h after CLP. Energy and lipid metabolism reduced significantly while FOXO1 levels increased remarkably in TECs during SAKI. The expressions of both AKT and CDK2 and the transcriptions of relevant mRNAs reduced significantly in TECs during SAKI while miR-21-3p expression increased remarkably. Both AKT and CDK2 were determined as the direct targets of miR-21-3p. Furthermore, by in vitro experiments, it was demonstrated that FOXO1 levels were regulated by miR-21-3p in TECs via AKT/CDK2 and AKT/CDK2-FOXO1 pathway was crucial in the regulations of miR-21-3p on lipid metabolism, cell cycle arrest, and apoptosis of TECs. Conclusions: MiR-21-3p mediates metabolism and cell fate alterations of TECs via manipulating AKT/CDK2-FOXO1 pathway, and that is crucial in the regulation of energy metabolism of TECs during SAKI.


Assuntos
Lesão Renal Aguda/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , MicroRNAs/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sepse/metabolismo , Transdução de Sinais , Lesão Renal Aguda/etiologia , Lesão Renal Aguda/patologia , Animais , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Túbulos Renais/metabolismo , Túbulos Renais/fisiologia , Ratos , Ratos Sprague-Dawley , Sepse/complicações , Sepse/patologia
12.
Acta Histochem ; 121(6): 695-703, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31239073

RESUMO

Renal fibrosis is a common mechanism that leads to all kidney diseases and Epithelial-mesenchymal transition (EMT) is considered as one of the potential mechanisms of renal fibrosis. Inhibitor of growth 4 (ING4) was reported to involve in several diseases; especially it was negatively correlated with lung fibrogenesis parameters. However, the role of ING4 and underlying mechanisms in EMT are still unknown. In this study, we used a UUO rat model to mimic renal fibrosis, which was examined by Masson and HE staining analysis. To explore the effects of ING4 on hypoxia-induced EMT, HK2 cells were treated with hypoxia to induce EMT and ING4 was over-expressed in hypoxia-treated HK2 cells by transfection of pEGFP-N1-ING4. MTT assay was used to describe the cell viability of HK2 cells under the hypoxic condition. The expression levels of ING4, hypoxia-inducible factor-1α (HIF-1α), and EMT markers (E-cadherin, N-cadherin and vimentin) were examined in vivo and in vitro by western blot, qRT-PCR, immunohistochemical staining or Immunofluorescence. Our results showed that, in a UUO rat model, ING4 was decreased and EMT was developed with reduction in E-cadherin and increase in N-cadherin and vimentin, suggesting a significant association between ING4 expression and EMT. Under hypoxia, E-cadherin was down-regulated and N-cadherin and vimentin were up-regulated, indicating that hypoxia induced EMT in HK2 cells. Nonetheless, changes in the expression of EMT biomarkers were inhibited by over-expression of ING4. Moreover, over-expressing ING4 decreased the expression of HIF-1α and snail in HK2 cells. These findings suggest that ING4 may inhibit hypoxia-induced EMT via decreasing HIF-1α and snail in HK2 cells, indicating the potential of ING4 as a therapeutic target for renal fibrosis.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Proteínas de Homeodomínio/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Túbulos Renais/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Hipóxia Celular , Linhagem Celular , Células Epiteliais/patologia , Fibrose , Humanos , Nefropatias/metabolismo , Nefropatias/patologia , Túbulos Renais/patologia , Ratos
13.
Eur J Pharmacol ; 855: 1-9, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31047876

RESUMO

Acute Kidney Injury (AKI) is associated with high morbidity and mortality. Ischemia and reperfusion (I/R) are events that lead to AKI through hypoxia, reactive oxygen species (ROS) production, oxidative stress and apoptosis. We aimed to evaluate the mechanism of nephroprotection mediated by Bisabolol in human tubular kidney cells after injury by I/R in vitro. HK2 cells were exposed to I/R and treated with Bisabolol. Cell viability was accessed by MTT assay. Cells were submitted to flow cytometry to evaluate necrotic/apoptotic cells, reactive oxygen species production and mitochondrial transmembrane depolarization. TBARS and GSH were used as parameters of redox balance. Also, KIM-1 supernatant levels were measured. In order to identify an interaction between bisabolol and NOX4, molecular docking and enzymatic assays were performed. Expression of isoform NOX4 on treated cells was examined by western-blot. Finally, cells were visualized by scanning electron microscopy. Bisabolol improved cell viability and prevented cell death by apoptosis, indicated also by the decreased levels of KIM-1. It was observed a decrease on reactive oxygen species production and mitochondrial depolarization, with antioxidant regulation by increased GSH and decreased lipid peroxidation. It was also demonstrated that bisabolol treatment can inhibit NOX4. Finally, SEM images showed that bisabolol reduced I/R-induced cell damage. Bisabolol treatment protects HK2 cells against oxidative damage occasioned by I/R. This effect is related to inhibition of apoptosis, decrease on KIM-1 release, reactive oxygen species accumulation and mitochondrial dysfunction. Bisabolol inhibited NOX4 activity in the tubular cells, impairing reactive oxygen species synthesis.


Assuntos
Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , NADPH Oxidases/metabolismo , Oxigênio/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Glutationa/metabolismo , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Humanos , Túbulos Renais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo
14.
EBioMedicine ; 43: 607-619, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31060900

RESUMO

BACKGROUND: The mitochondrial associated endoplasmic reticulum (ER) membrane (MAM) provides a platform for communication between the mitochondria and ER, and it plays a vital role in many biological functions. Disulphide-bond A oxidoreductase-like protein (DsbA-L), expressed in the MAM, serves as an antioxidant and reduces ER stress. However, the role of DsbA-L and MAM in kidney pathobiology remains unclear. METHODS: Molecular biology techniques, transmission electron microscopy (TEM), in situ proximity ligation assays (PLAs), confocal microscopy, TUNEL staining and flow cytometry were utilized to analyse apoptosis and status of MAM in DsbA-L mutant mice. FINDINGS: We showed that MAM was significantly reduced in the kidneys of streptozotocin-induced diabetic mice, which correlated with the extent of renal injury. We also observed a correlation between the loss of MAM integrity and increased apoptosis and renal injury in diabetic nephropathy (DN). These alterations were further exacerbated in diabetic DsbA-L gene-deficient mice (DsbA-L-/-). In vitro, overexpression of DsbA-L in HK-2 cells restored MAM integrity and reduced apoptosis induced by high-glucose ambience. These beneficial effects were partially blocked by overexpression of FATE-1, a MAM uncoupling protein. Finally, the expression of DsbA-L was positively correlated with MAM integrity in the kidneys of DN patients but negatively correlated with apoptosis and renal injury. INTERPRETATION: Our results indicate that DsbA-L exerts an antiapoptotic effect by maintaining MAM integrity, which is apparently disrupted in DN. FUND: This work was supported by the National Natural Science Foundation of China (81730018), the National Key R&D Program of China (2016YFC1305501) and NIH (DK60635).


Assuntos
Retículo Endoplasmático/metabolismo , Glucose/metabolismo , Glutationa Transferase/metabolismo , Membranas Intracelulares/metabolismo , Túbulos Renais/metabolismo , Animais , Biomarcadores , Glicemia , Linhagem Celular , Diabetes Mellitus Experimental , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Feminino , Túbulos Renais/patologia , Túbulos Renais/ultraestrutura , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo
15.
Toxicol Lett ; 311: 27-36, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31039415

RESUMO

Methotrexate (MTX) is a derivate of folic acid, commonly used as an anchor drug for the treatment and management of malignant diseases and autoimmune disorders. However, nephrotoxicity is an important drawback of MTX therapy. Unfortunately, there are not enough studies reporting the nature of the renal failure induced by MTX. Thus, the aim of this study was to evaluate the time course of renal handling of water and electrolytes in male Wistar rats, after the exposure to a unique dose of MTX (80 mg/kg b.w.). Experiments were carried out at day 2, day 4, day 8 and day 14 after MTX administration. Several parameters of kidney function related to water and electrolytes handling were evaluated. Renal expression and urinary excretion of aquaporin-2 (AQP2) and Na-K-2Cl-cotransporter (NKCC2) were determined by Western blotting. MTX produced alterations on water handling on the second day after treatment, showing a significant increase in solute free water reabsorption which might be mediated by the increased expression of AQP2 in apical membranes. On the other hand, MTX produced alterations on electrolytes handling on the fourth day after treatment, showing a significant decrease of sodium chloride excretion, mediated at least in part, by the increase renal expression of NKCC2. These results provide valuable information to clinical practice in order to be able to find therapeutic targets that diminish adverse effects and health deterioration. Moreover, MTX treatment altered AQP2 and NKCC2 urinary excretion allowing postulating these transporters as potential biomarkers of MTX induced nephrotoxicity.


Assuntos
Aquaporina 2/metabolismo , Eletrólitos/metabolismo , Nefropatias/induzido quimicamente , Túbulos Renais/efeitos dos fármacos , Metotrexato/toxicidade , Reabsorção Renal/efeitos dos fármacos , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Água/metabolismo , Animais , Biomarcadores/metabolismo , Cloretos/metabolismo , Nefropatias/metabolismo , Túbulos Renais/metabolismo , Masculino , Potássio/metabolismo , Ratos Wistar , Sódio/metabolismo , Fatores de Tempo , Urodinâmica/efeitos dos fármacos
16.
Nat Immunol ; 20(7): 915-927, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31110316

RESUMO

The molecular and cellular processes that lead to renal damage and to the heterogeneity of lupus nephritis (LN) are not well understood. We applied single-cell RNA sequencing (scRNA-seq) to renal biopsies from patients with LN and evaluated skin biopsies as a potential source of diagnostic and prognostic markers of renal disease. Type I interferon (IFN)-response signatures in tubular cells and keratinocytes distinguished patients with LN from healthy control subjects. Moreover, a high IFN-response signature and fibrotic signature in tubular cells were each associated with failure to respond to treatment. Analysis of tubular cells from patients with proliferative, membranous and mixed LN indicated pathways relevant to inflammation and fibrosis, which offer insight into their histologic differences. In summary, we applied scRNA-seq to LN to deconstruct its heterogeneity and identify novel targets for personalized approaches to therapy.


Assuntos
Perfilação da Expressão Gênica , Interferon Tipo I/metabolismo , Queratinócitos/metabolismo , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Nefrite Lúpica/genética , Nefrite Lúpica/metabolismo , Transcriptoma , Biópsia , Linhagem da Célula/genética , Biologia Computacional/métodos , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Fibrose , Perfilação da Expressão Gênica/métodos , Humanos , Nefrite Lúpica/patologia , Ligação Proteica , Transdução de Sinais , Análise de Célula Única , Pele/imunologia , Pele/metabolismo , Pele/patologia
17.
Cell Physiol Biochem ; 52(6): 1463-1483, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31099507

RESUMO

BACKGROUND/AIMS: The therapeutic potential of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) in kidney injury has been largely reported. However, new approaches are necessary to optimize the efficacy in the treatment of renal diseases. MSCs physiologically are under a low O2 partial pressure (pO2), and culturing adipose-derived MSCs (ADMSCs) in hypoxia alters their secretory paracrine properties. The aim of this study was to evaluate whether hypoxia preconditioning of ADMSCs alters the properties of secreted EVs to improve renal recovery after ischemia-reperfusion injury (IRI). METHODS: The supernatants of ADMSCs cultivated under 21% pO2 (control) or 1% pO2 (hypoxia) were ultracentrifuged for EVs isolation that were posteriorly characterized by flow cytometry and electron microscopy. The uptake and effects of these EVs were analyzed by using in vitro and in vivo models. HK-2 renal tubule cell line was submitted do ATP depletion injury model. Proteomic analyses of these cells treated with EVs after injury were performed by nano-UPLC tandem nano-ESI-HDMSE method. For in vivo analyses, male Wistar rats were submitted to 45 min bilateral ischemia, followed by renal intracapsular administration of ADMSC-EVs within a 72 h reperfusion period. Histological, immunohistochemical and qRT-PCR analysis of these kidneys were performed to evaluate cell death, inflammation and oxidative stress. Kidney function was evaluated by measuring the blood levels of creatinine and urea. RESULTS: The results demonstrate that hypoxia increases the ADMSCs capacity to secrete EVs that trigger different energy supply, antiapoptotic, immunomodulatory, angiogenic and anti-oxidative stress responses in renal tissue compared with EVs secreted in normoxia. Proteomic analyses of renal tubule cells treated with EVs from ADMSCs in normoxia and hypoxia give a specific signature of modulated proteins for each type of EVs, indicating regulation of distinct biological processes. CONCLUSION: In summary, hypoxia potentially offers an interesting strategy to enhance the properties of EVs in the treatment of acute kidney disease.


Assuntos
Lesão Renal Aguda/terapia , Vesículas Extracelulares/transplante , Células-Tronco Mesenquimais/metabolismo , Traumatismo por Reperfusão/terapia , Lesão Renal Aguda/metabolismo , Lesão Renal Aguda/patologia , Tecido Adiposo/citologia , Animais , Hipóxia Celular , Linhagem Celular , Células Cultivadas , Vesículas Extracelulares/metabolismo , Humanos , Rim/metabolismo , Rim/patologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Masculino , Células-Tronco Mesenquimais/citologia , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia
18.
Transplant Proc ; 51(5): 1343-1347, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31076149

RESUMO

BACKGROUND: Lipofuscin is an indicator of aging. We examined the clinicopathologic significance of lipofuscin deposition in the renal tubules of renal allografts. METHOD: We analyzed allograft biopsy specimens from living kidney transplantations from January to December 2015. For controls, we analyzed native kidney biopsy specimens obtained from January 2015 to December 2016. We identified granules with a yellow-to-tan shade in renal tubules as lipofuscin. RESULTS: The donor age at transplantation was significantly older in lipofuscin deposition biopsy specimens than in those without, whereas the time after transplantation age was not different between the 2 groups with renal allografts. In native kidney biopsies, age at biopsy was significantly older in lipofuscin deposition biopsy specimens than in those without. We compared "massive lipofuscin deposition," defined as lipofuscin deposition on both sides of 3 or more renal tubules, and donor-age matched control allograft biopsies without lipofuscin deposition. Comparing these 2 groups, recipient age at transplantation was significantly older in the massive lipofuscin deposition group. CONCLUSION: Lipofuscin deposition on tubular epithelium is not a surrogate marker of aging of kidneys allografts, although lipofuscin deposition was significantly greater in older tissues from native kidneys. The older age of recipients may be associated with massive lipofuscin deposition in renal allografts.


Assuntos
Transplante de Rim , Túbulos Renais/patologia , Lipofuscina/análise , Adulto , Idoso , Aloenxertos , Biomarcadores , Feminino , Humanos , Túbulos Renais/metabolismo , Lipofuscina/metabolismo , Masculino , Pessoa de Meia-Idade , Transplante Homólogo
19.
Am J Physiol Renal Physiol ; 316(6): F1273-F1281, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31017010

RESUMO

Acute kidney injury (AKI) initiated by sepsis remains a thorny problem despite recent advancements in its clinical management. Having been found to be activated during AKI, fibroblast growth factor-inducible molecule 14 (Fn14) may be a potential therapeutic target because of its involvement in the molecular basis of injury. Here, we report that LPS induces apoptosis of mouse cortical tubule cells mediated by Fn14, for which simultaneous Toll-like receptor (TLR)4 activation is required. Mechanistically, TLR4 activation by lipopolysaccharide, through disassociating E3 ligase SCFFbxw7α from Fn14, dismantles Lys48-linked polyubiquitination of Fn14 and stabilizes it. Pharmacological deactivation of Fn14 with monoclonal antibody ITEM-2 provides effective protection against lethal sepsis and AKI in mice. Our study underscores an adaptive mechanism whereby TLR4 regulates SCFFbxw7α-dependent Fn14 stabilization during inflammatory tubular damage and further supports investigation of targeting Fn14 in clinical trials of patients with septic AKI.


Assuntos
Lesão Renal Aguda/metabolismo , Proteína 7 com Repetições F-Box-WD/metabolismo , Túbulos Renais/metabolismo , Macrófagos/metabolismo , Sepse/complicações , Receptor de TWEAK/metabolismo , Lesão Renal Aguda/genética , Lesão Renal Aguda/microbiologia , Lesão Renal Aguda/patologia , Animais , Apoptose , Modelos Animais de Doenças , Proteína 7 com Repetições F-Box-WD/genética , Túbulos Renais/microbiologia , Túbulos Renais/patologia , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Estabilidade Proteica , Células RAW 264.7 , Sepse/microbiologia , Transdução de Sinais , Receptor de TWEAK/genética , Receptor 4 Toll-Like/metabolismo
20.
Life Sci ; 228: 1-10, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31028803

RESUMO

AIMS: This study aimed to investigate the role of transforming growth factor-ß-activated protein kinase 1(TAK1) in the development of diabetic nephropathy (DN) by regulating the protein stability of Ski-related novel protein N(SnoN). MAIN METHODS: A combination of in vivo and in vitro model systems was used to investigate how TAK1 regulated the expression of SnoN protein in DN. The study determined the effects of modulating the expression or activity of TAK1 on the SnoN protein level and its influence on the epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) deposition. KEY FINDINGS: Under the high-glucose condition, the activation of TGF-ß1/TAK1-induced phosphorylation and ubiquitination of SnoN protein resulted in reduced SnoN protein level as a consequence of enhanced SnoN degradation, which promoted EMT and ECM deposition in renal tubular epithelial cells. The study showed that TAK1 impaired SnoN protein level by decreasing the protein stability of SnoN. SIGNIFICANCE: TAK1 mediated the phosphorylation of SnoN, resulting in SnoN ubiquitination and eventual degradation, which enhanced EMT and ECM deposition to promote renal fibrosis during DN.


Assuntos
Nefropatias Diabéticas/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Nefropatias Diabéticas/patologia , Transição Epitelial-Mesenquimal , Fibrose , Glucose/metabolismo , Rim/metabolismo , Rim/patologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Masculino , Fosforilação , Estabilidade Proteica , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta1/metabolismo , Ubiquitinação
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