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1.
Life Sci ; 271: 119173, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33556375

RESUMO

AIMS: Cell cycle arrest plays critical roles in preventing renal tubular epithelial cell (RTEC) injury and maladaptation after the onset of chronic kidney disease (CKD), but the underlying mechanism governing this arrest has not been fully elucidated. This study was designed to determine the underlying role of YB-1 in promoting cell cycle progression and nuclear translocation in HK-2 cells induced by trimethylamine N-oxide (TMAO). MAIN METHODS: YB-1 primarily accumulated in the cytoplasm in HK-2 cells after they were treated with TMAO for 30 min and 6 h. Gene expression was analysed using RNA sequencing in HK-2 cells treated with TMAO. Cell cycle progression was analysed via flow cytometry. Luciferase assay and ChIP-PCR were performed to determine the relationship between transcription factor YB-1 and Gadd45a promoter region. Additionally, mice were fed with TMAO to test renal dysfunction and measure the expression of YB-1, GADD45a and CCNA2 in the kidney sections through immunohistochemistry. KEY FINDINGS: YB-1 primarily accumulated in the cytoplasm in HK-2 cells after they were treated with TMAO for 30 min and 6 h. RNA sequencing analysis showed that the cell cycle checkpoint genes growth arrest and DNA damage (Gadd)45a, Gadd45g, cyclin (Ccn)a2, Ccnb1, Ccne1 and Ccnf were differentially expressed in HK-2 cells after treated with 400 µM TMAO for 30 min. Flow cytometry results demonstrated that cell cycle progression was blocked at the G2/M checkpoint. In animal models, elevated dietary TMAO directly led to progressive renal tubulointerstitial dysfunction and inhibited the expression of YB-1 in kidney. Moreover, YB-1 was determined to regulate Gadd45a expression by directly binding to its promoter region. YB-1 expression was negatively correlated with the expression of Gadd45a and Gadd45g but positively correlated with Ccna2, Ccnb1, Ccne1 and Ccnf in CKD. SIGNIFICANCE: YB-1 may be a reliable molecular target and an effective prognostic biomarker for CKD.


Assuntos
Proteínas de Ciclo Celular/biossíntese , Ciclo Celular/fisiologia , Regulação para Baixo/fisiologia , Metilaminas/toxicidade , Insuficiência Renal Crônica/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Expressão Gênica , Humanos , Túbulos Renais/citologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Camundongos Endogâmicos C57BL , Insuficiência Renal Crônica/induzido quimicamente , Insuficiência Renal Crônica/genética
3.
Artigo em Japonês | MEDLINE | ID: mdl-33342936

RESUMO

Renal tubular cell death is caused by various extracellular stresses including toxic amounts of cadmium, an occupational and environmental pollutant metal, and is responsible for renal dysfunction. While cadmium exposure disrupts many intracellular signaling pathways, the molecular mechanism underlying cadmium-induced renal tubular cell death has not yet been fully elucidated. We have recently identified two important intracellular signaling pathways that promote cadmium-induced renal tubular cell death: the Notch1 signaling and activin receptor-like kinase (ALK) 4/5 signaling (also known as the activin-transforming growth factor ß receptor pathways). In this review paper, we introduce our previous experimental findings, focusing on Notch1 and ALK4/5 signaling pathways, which may uncover the molecular mechanisms involved in cadmium-induced renal tubular cell death.


Assuntos
Receptores de Ativinas Tipo I/metabolismo , Cádmio/toxicidade , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Poluentes Ambientais/toxicidade , Túbulos Renais/citologia , Túbulos Renais/patologia , Exposição Ocupacional/efeitos adversos , Receptor Notch1/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Animais , Humanos , Camundongos , Ratos
4.
Life Sci ; 261: 118484, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32976885

RESUMO

AIM: Chloride channel 7 (CLC-7), broadly expressed in kidney tissues, affects the lysosome degradation pathway. And redox status impairment contributes to cell apoptosis and activates autophagy flux. This study mainly investigates the role and molecular mechanism of CLC-7 in redox status impairment-induced autophagic flux and apoptosis. MAIN METHODS: When NRK52E cells, rat renal tubular epithelial cells, were exposed to H2O2 treatment, apoptosis, autophagy flux, and CLC-7 expression were detected. Further investigation was done to observe the change of apoptosis and autophagy flux in renal cells under overexpression or knocking down of CLC-7. The lysosomes acidity, lysosome enzyme Cathepsin D activity and phosphorylation of Ampk/mTOR were also examined when CLC-7 was overexpressed or knocked down. KEY FINDINGS: Redox status impairment induced apoptosis and autophagy flux in NRK52E cells and upregulated CLC-7. Overexpression of CLC-7 increased lysosome acidity and Cathepsin D activity. In cells with CLC-7 overexpression, we observed a significant increase of autophagy flux and decline of apoptosis, as well as an apparent increase of p-Ampk and decrease of p-mTOR. On the contrary, cells with knocking down CLC-7 led to opposite results. SIGNIFICANCES: CLC-7 is essential to maintain and enhance acidity and enzyme activity in lysosome. Through activating autophagy flux, it exerts survival against renal tubular epithelial cell apoptosis induced by redox status impairment. Its function to modulate Ampk/mTOR pathway is the possible reason why CLC-7 can trigger autophagy flux.


Assuntos
Autofagia , Canais de Cloreto/metabolismo , Células Epiteliais/citologia , Túbulos Renais/citologia , Animais , Apoptose , Linhagem Celular , Células Epiteliais/metabolismo , Peróxido de Hidrogênio/metabolismo , Túbulos Renais/metabolismo , Oxirredução , Ratos
5.
Nat Commun ; 11(1): 3383, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636391

RESUMO

The endogenous repair process can result in recovery after acute kidney injury (AKI) with adaptive proliferation of tubular epithelial cells, but repair can also lead to fibrosis and progressive kidney disease. There is currently limited knowledge about transcriptional regulators regulating these repair programs. Herein we establish the enhancer and super-enhancer landscape after AKI by ChIP-seq in uninjured and repairing kidneys on day two after ischemia reperfusion injury (IRI). We identify key transcription factors including HNF4A, GR, STAT3 and STAT5, which show specific binding at enhancer and super-enhancer sites, revealing enhancer dynamics and transcriptional changes during kidney repair. Loss of bromodomain-containing protein 4 function before IRI leads to impaired recovery after AKI and increased mortality. Our comprehensive analysis of epigenetic changes after kidney injury in vivo has the potential to identify targets for therapeutic intervention. Importantly, our data also call attention to potential caveats involved in use of BET inhibitors in patients at risk for AKI.


Assuntos
Lesão Renal Aguda/genética , Elementos Facilitadores Genéticos , Túbulos Renais/citologia , Lesão Renal Aguda/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Proliferação de Células , Epigênese Genética , Fibrose , Fator 4 Nuclear de Hepatócito/metabolismo , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Nucleares , Receptores de Glucocorticoides/metabolismo , Elementos Reguladores de Transcrição , Traumatismo por Reperfusão/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Fatores de Transcrição , Transcrição Genética
6.
J Biol Regul Homeost Agents ; 34(1): 69-82, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32392921

RESUMO

Baicalin has been used in China to treat inflammation-related diseases, such as inflammation-induced acute kidney injury (AKI). However, the specific mechanism of baicalin remains unclear. To observe the protective effects of baicalin on lipopolysaccharide (LPS)-induced inflammatory injury of renal tubular epithelial cells (HK-2 cells) and to explore its protective mechanism. LPS (1 mg/L) was used to induce an HK-2 cell inflammatory injury model in vitro. The cells were divided into seven groups: the normal control group, LPS-induced group, LPS plus 5 µmol/L baicalin treatment group, LPS plus 15 µmol/L baicalin treatment group, LPS plus 25 µmol/L baicalin treatment group, LPS plus 50 µmol/L baicalin treatment group, and LPS plus 75 µmol/L baicalin treatment group. 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-H-tetrazolium bromide (MTT) assay was employed for detecting the relative survival rate of HK-2 cells. Enzyme-linked immunosorbent assay was used for detecting the levels of inflammatory factors, including interleukin-6 (IL-6), IL-1ß, and tumor necrosis factor-α (TNF-α). Moreover, the expression of inducible nitric oxide synthase (iNOS); cyclooxygenase-2 (COX-2); nuclear factor kB65 (NF-κB65); phosphorylated NF-κB inhibitory protein-α (p-IκB-α); NF-κB inhibitory protein (IκB); human thioredoxin interacting protein (TXNIP); and human NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) were determined by Western blot analysis. The expression levels of NLRP3 and TXNIP mRNA and miR-223-3p were determined by RT-PCR. Results found that the relative survival rate of HK-2 cells treated with different baicalin concentrations was significantly increased (P<0.05) and the levels of the inflammatory factors IL-6, IL-1ß, and TNF-α were significantly decreased (P<0.05) compared with those of the LPS-induced group. The expression levels of the inflammatory proteins inducible nitric oxide synthase and cyclooxygenase-2 and the genes expressions of TXNIP and NLRP3 were significantly decreased in the cells (P<0.05), while the expression level of miR-223- 3p was significantly increased (P<0.05). These changes were induced in a dose-dependent manner. The results suggest that baicalin significantly inhibited the expression of inflammation-related proteins and alleviated LPS-induced inflammatory injury in HK-2 cells. The mechanism may be associated with the inhibition of activation of the TXNIP/NLRP3 inflammatory pathway, which might be mediated by increased expression of miR-223-3p. Thus, NLRP3 is a regulatory target of miR-223-3p.


Assuntos
Proteínas de Transporte/metabolismo , Células Epiteliais/efeitos dos fármacos , Flavonoides/farmacologia , MicroRNAs/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Transdução de Sinais , Células Cultivadas , Citocinas/metabolismo , Humanos , Inflamação , Túbulos Renais/citologia , Lipopolissacarídeos
7.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(3): 212-219, 2020 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-32389168

RESUMO

Objective To investigate the effect of enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) on the development of renal tubular epithelial-mesenchymal transformation (EMT) and diabetic nephropathy (DN). Methods Mouse model of type 1 diabetic nephropathy (T1DN) was established by intraperitoneal injection of streptozotocin (STZ) (55 mg/kg) and randomly divided into normal control and diabetic group. The mice were killed, and their biochemical indexes (blood glucose, creatinine, microalbumin and total protein in urine) of blood and urine were recorded. The kidneys were subjected to HE and Masson staining to observe morphological changes. Immunohistochemical staining was used to observe the expression and localization of type IV collagen (Col4) and EZH2. The mRNA and protein expressions of E-cadherin, α-smooth muscle actin (α-SMA), Col4 and EZH2 were detected by real-time fluorescent quantitative PCR and Western blotting. Rat renal tubular epithelial cells were cultured with high glucose and transfected with small interfering RNA (siRNA) of EZH2.The protein levels of E-cadherin, α-SMA, Col4 and EZH2 were detected by Western blot analysis. Results Compared with the normal group, blood glucose, serum creatinine, microalbuminuria and total urine protein significantly increased in the diabetic group. The mRNA and protein levels of E-cadherin, α-SMA, Col4 and EZH2 went up; the tubular lumen collapsed; the basement membrane of glomerulus thickened and there was a large amount of collagen deposition in the renal interstitium. Compared with normal sugar, high glucose stimulation promoted EMT and significantly up-regulated EZH2 expression. Compared with high glucose group, the transfection of EZH2 siRNA in high glucose inhibited EMT. Conclusion EZH2 can effectively promote the EMT process of renal tubular epithelial cells and may participate in the occurrence and development of DN through this role.


Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas/patologia , Proteína Potenciadora do Homólogo 2 de Zeste/fisiologia , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Animais , Progressão da Doença , Túbulos Renais/citologia , Camundongos , Distribuição Aleatória
8.
J Biol Regul Homeost Agents ; 34(1): 57-67, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32466632

RESUMO

Klotho is a putative aging suppressor gene that is primarily expressed in renal tubular epithelial cells. Its expression has been reported to protect against fibrosis in human chronic kidney disease. However, the roles of klotho in epithelial-mesenchymal transition (EMT) and renal fibrosis are yet to be elucidated. The present study aimed to investigate the putative roles of klotho in angiotensin (Ang) II-induced damage of renal tubular epithelial cells. NRK-52E rat cells were treated with various combinations of Ang II, the Ang-converting enzyme inhibitor fosinopril (Fos) and the Ang II receptor antagonist valsartan (Val). The levels of transforming growth factor (TGF)-ß1, soluble klotho, α-smooth muscle actin (α-SMA) and E-cadherin in NRK-52E culture supernatants were measured using enzyme-linked immunosorbent assays. Furthermore, the mRNA and protein expression of TGF-ß1, klotho, α-SMA and E-cadherin was detected using semiquantitative reverse transcription-polymerase chain reaction, immunocytochemistry and Western blot analysis. The results demonstrated that Ang II inhibited the expression of klotho and E-cadherin, while it upregulated the expression of TGF-ß1 and α-SMA, in NRK52E cells. Fos and/or Val were revealed to enhance klotho and E-cadherin expression, and suppress the expression of TGF-ß1 and α-SMA, compared with the Ang II-only group. Furthermore, a positive linear correlation was detected between the expression of klotho and E-cadherin, while negative linear correlations with klotho expression were detected for TGF-ß1 and α-SMA expression. In conclusion, the expression of klotho was demonstrated to be enhanced following treatment with Fos and Val in Ang II-treated NRK-52E cells. The present results indicate that klotho may be involved in the inhibition of Ang II-induced EMT in renal tubular epithelial cells. Therefore, klotho may serve as a protective factor in renal tubulointerstitial fibrosis and aid the treatment of chronic kidney disease (CKD) patients using precision therapy.


Assuntos
Células Epiteliais/citologia , Transição Epitelial-Mesenquimal , Glucuronidase/fisiologia , Sistema Renina-Angiotensina , Actinas/metabolismo , Angiotensina II/farmacologia , Animais , Caderinas/metabolismo , Linhagem Celular , Fibrose , Fosinopril/farmacologia , Humanos , Túbulos Renais/citologia , Ratos , Fator de Crescimento Transformador beta1/metabolismo , Valsartana/farmacologia
9.
Lab Invest ; 100(9): 1184-1196, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32366943

RESUMO

Acute kidney injury triggers a complex cascade of molecular responses that can culminate in maladaptive repair and fibrosis. We have previously reported that the matrix protein thrombospondin-1 (TSP1), binding its high affinity its receptor CD47, promotes acute kidney injury. However, the role of this pathway in promoting fibrosis is less clear. Hypothesizing that limiting TSP1-CD47 signaling is protective against fibrosis, we interrogated this pathway in a mouse model of chronic ischemic kidney injury. Plasma and renal parenchymal expression of TSP1 in patients with chronic kidney disease was also assessed. We found that CD47-/- mice or wild-type mice treated with a CD47 blocking antibody showed clear amelioration of fibrotic histological changes compared to control animals. Wild-type mice showed upregulated TSP1 and pro-fibrotic markers which were significantly abrogated in CD47-/- and antibody-treated cohorts. Renal tubular epithelial cells isolated from WT mice showed robust upregulation of pro-fibrotic markers following hypoxic stress or exogenous TSP1, which was mitigated in CD47-/- cells. Patient sera showed a proportionate correlation between TSP1 levels and worsening glomerular filtration rate. Immunohistochemistry of human kidney tissue demonstrated tubular and glomerular matrix localization of TSP1 expression in patients with CKD. These data suggest that renal tubular epithelial cells contribute to fibrosis by activating TSP1-CD47 signaling, and point to CD47 as a potential target to limit fibrosis following ischemic injury.


Assuntos
Antígeno CD47/metabolismo , Rim/metabolismo , Transdução de Sinais , Trombospondina 1/metabolismo , Animais , Antígeno CD47/genética , Células Cultivadas , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Fibrose , Humanos , Isquemia , Rim/irrigação sanguínea , Rim/patologia , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout
10.
PLoS One ; 15(4): e0232055, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32324796

RESUMO

Chronic kidney diseases affect more than 800 million people globally and remain a high unmet need. Various therapeutic targets are currently under evaluation in pre-clinical and clinical studies. Because the growth arrest specific gene 6 (Gas6)/AXL pathway has been implicated in the pathogenesis of kidney diseases, we generated a novel selective and potent AXL inhibitor, CH5451098, and we evaluated its efficacy and elucidated its mechanism in an NEP25 mouse model that follows the clinical course of glomerular nephritis. In this model, CH5451098 significantly ameliorated the excretion of urinary albumin and elevation of serum creatinine. Additionally, it also inhibited tubulointerstitial fibrosis and tubular damage. To elucidate the mechanism behind these changes, we analyzed the effect of CH5451098 against transforming growth factor ß1 (TGFß1) and Gas6, which is a ligand of AXL receptor, in NRK-52E renal tubular epithelial cells. CH5451098 inhibited epithelial-to-mesenchymal transition (EMT) caused by the synergistic effects of TGFß1 and Gas6 in NRK-52E cells. This inhibition was also observed in NEP25 mice. Taken together, these results suggest that CH5451098 could ameliorate kidney dysfunction in glomerular nephritis by inhibiting EMT in tubular cells. These results reveal that AXL strongly contributes to the disease progression of glomerular nephritis.


Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glomerulonefrite/tratamento farmacológico , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Túbulos Renais/citologia , Rim/fisiopatologia , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Albuminas/análise , Animais , Linhagem Celular , Creatinina/sangue , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Glomerulonefrite/genética , Glomerulonefrite/metabolismo , Glomerulonefrite/fisiopatologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Rim/efeitos dos fármacos , Testes de Função Renal , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Camundongos , Camundongos Transgênicos , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Ratos , Fator de Crescimento Transformador beta1/genética
11.
PLoS One ; 15(3): e0228385, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32134954

RESUMO

Our earlier studies proved that RIPK3-mediated necroptosis might be an important mode of renal tubular cell death in rats with chronic renal injury and the necroptotic cell death can be triggered by tumor necrosis factor-α (TNF-α) in vitro, but the triggering role of angiotensin II (AngII), which exerts notable effects on renal cells for the initiation and progression of renal tubulointerstitial fibrosis, is largely unknown. Here, we identified the presence of necroptotic cell death in the tubular cells of AngII-induced chronic renal injury and fibrosis mice and assessed the percentage of necroptotic renal tubular cell death with the disruption of this necroptosis by the addition of necrostatin-1 (Nec-1). Furthermore, the observation was further confirmed in HK-2 cells treated with AngII and RIPK1/3 or MLKL inhibitors. The detection of Fas and FasL proteins led us to investigate the contribution of the Fas/FasL signaling pathway to AngII-induced necroptosis. Disruption of FasL decreased the percentage of necroptotic cells, suggesting that Fas and FasL are likely key signal molecules in the necroptosis of HK-2 cells induced by AngII. Our data suggest that AngII exposure might trigger RIPK3-MLKL-mediated necroptosis in renal tubular epithelial cells by activating the Fas/FasL signaling pathway in vivo and in vitro.


Assuntos
Angiotensina II/farmacologia , Proteína Ligante Fas/metabolismo , Túbulos Renais/citologia , Necroptose/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptor fas/metabolismo , Animais , Linhagem Celular , Fibrose , Humanos , Túbulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos
12.
Mediators Inflamm ; 2020: 6357046, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32089648

RESUMO

Inflammation is a major contributor to tubular epithelium injury in kidney disorders, and the involvement of blood platelets in driving inflammation is increasingly stressed. CD154, the ligand of CD40, is one of the mediators supporting platelet proinflammatory properties. Although hypoxia is an essential constituent of the inflammatory reaction, if and how platelets and CD154 regulate inflammation in hypoxic conditions remain unclear. Here, we studied the control by CD154 of the proinflammatory cytokine interleukin- (IL-) 6 secretion in short-term oxygen (O2) deprivation conditions, using the HK-2 cell line as a kidney tubular epithelial cell (TEC) model. IL-6 secretion was markedly stimulated by CD154 after 1 to 3 hours of hypoxic stress. Both intracellular IL-6 expression and secretion were stimulated by CD154 and associated with a strong upregulation of IL-6 mRNA and increased transcription. Searching for inhibitors of CD154-mediated IL-6 production by HK-2 cells in hypoxic conditions, we observed that chloroquine, a drug that has been repurposed as an anti-inflammatory agent, alleviated this induction. Therefore, CD154 is a potent early stimulus for IL-6 secretion by TECs in O2 deprivation conditions, a mechanism likely to take part in the deleterious inflammatory consequences of platelet activation in kidney tubular injury. The inhibition of CD154-induced IL-6 production by chloroquine suggests the potential usefulness of this drug as a therapeutic adjunct in conditions associated with acute kidney injury.


Assuntos
Ligante de CD40/farmacologia , Hipóxia Celular/fisiologia , Cloroquina/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Interleucina-6/metabolismo , Túbulos Renais/citologia , Apoptose , Western Blotting , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Microscopia de Fluorescência , Reação em Cadeia da Polimerase em Tempo Real
13.
Am J Physiol Renal Physiol ; 318(3): F793-F803, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32036696

RESUMO

Acute kidney injury has a high global morbidity associated with an increased risk of death and chronic kidney disease. Renal tubular epithelial cell regeneration following injury may be a decisive factor in renal repair or the progression of acute kidney injury to chronic kidney disease, but the underlying mechanism of abnormal renal tubular repair remains unclear. In the present study, we investigated the role of heterotrimeric G stimulatory protein α-subunit (Gsa) in renal tubular epithelial cell regeneration. We generated renal tubule epithelium-specific Gsa knockout (GsaKspKO) mice to show the essential role of Gsa in renal tubular epithelial cell regeneration in two AKI models: acute aristolochic acid nephropathy (AAN) and unilateral ischemia-reperfusion injury (UIRI). GsaKspKO mice developed more severe renal impairment after AAN and UIRI, higher serum creatinine levels, and more substantial tubular necrosis than wild-type mice. More importantly, Gsa inactivation impaired renal tubular epithelial cell proliferation by reducing bromodeoxyuridine+ cell numbers in the AAN model and inhibiting cyclin-dependent kinase 2/cyclin E1 expression in the UIRI model. This reduced proliferation was further supported in vitro with Gsa-targeting siRNA. Downregulation of Gsa inhibited tubular epithelial cell proliferation in HK-2 and mIMCD-3 cells. Furthermore, Gsa downregulation inhibited cyclin-dependent kinase 2/cyclin E1 expression, which was dependent on the Raf-MEK-ERK signaling pathway. In conclusion, Gsa is required for tubular epithelial cell regeneration during kidney repair after AKI. Loss of Gsa impairs renal tubular epithelial cell regeneration by blocking the Raf-MEK-ERK pathway.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Nefropatias/etiologia , Animais , Ácidos Aristolóquicos , Linhagem Celular , Proliferação de Células , Proteínas Quinases Dependentes de AMP Cíclico/genética , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Regulação para Baixo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Nefropatias/metabolismo , Túbulos Renais/citologia , Camundongos , Camundongos Knockout , Traumatismo por Reperfusão
14.
Oxid Med Cell Longev ; 2020: 8708236, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32104542

RESUMO

Senescence of renal tubular epithelial cells plays an important role in diabetic nephropathy, but the mechanism is unknown. Metformin may alleviate diabetic nephropathy by reducing this senescence. This study is aimed at clarifying the effects and mechanism of metformin on the senescence of renal tubular epithelial cells in diabetic nephropathy. We found that metformin reduced the expression of senescence-associated gene P21 in high-glucose-induced (30 mmol/L) renal tubular epithelial cells and decreased the ß-galactosidase positive staining rate (decreased 16%, p < 0.01). Metformin was able to reduce senescence by upregulating the expression of RNA-binding protein MBNL1 and miR-130a-3p and reducing STAT3 expression. MBNL1 prolonged the half-life of miR-130a-3p, and miR-130a-3p could negatively regulate STAT3 by binding to its mRNA 3'UTR. In db/db diabetic mice, we found an enhanced senescence level combined with low expression of MBNL1 and miR-130a-3p and high expression of STAT3 compared with db/m control mice during nephropathy development. Meanwhile, metformin (200 mg/kg/day) could increase the expression of MBNL1 and miR-130a-3p and decreased STAT3 expression, thus reducing this senescence in db/db mice. Our results suggest that metformin reduces the senescence of renal tubular epithelial cells in diabetic nephropathy via the MBNL1/miR-130a-3p/STAT3 pathway, which provided new ideas for the therapy of this disease.


Assuntos
Senescência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Nefropatias Diabéticas/tratamento farmacológico , Metformina/uso terapêutico , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Túbulos Renais/citologia , Camundongos , MicroRNAs/genética , Proteínas de Ligação a RNA/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais/efeitos dos fármacos
15.
Proc Natl Acad Sci U S A ; 117(3): 1779-1787, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31907321

RESUMO

Insects are highly successful, in part through an excellent ability to osmoregulate. The renal (Malpighian) tubules can secrete fluid faster on a per-cell basis than any other epithelium, but the route for these remarkable water fluxes has not been established. In Drosophila melanogaster, we show that 4 genes of the major intrinsic protein family are expressed at a very high level in the fly renal tissue: the aquaporins (AQPs) Drip and Prip and the aquaglyceroporins Eglp2 and Eglp4 As predicted from their structure, and by their transport function by expressing these proteins in Xenopus oocytes, Drip, Prip, and Eglp2 show significant and specific water permeability, whereas Eglp2 and Eglp4 show very high permeability to glycerol and urea. Knockdowns of any of these genes result in impaired hormone-induced fluid secretion. The Drosophila tubule has 2 main secretory cell types: active cation-transporting principal cells, wherein the aquaglyceroporins localize to opposite plasma membranes, and small stellate cells, the site of the chloride shunt conductance, with these AQPs localizing to opposite plasma membranes. This suggests a model in which osmotically obliged water flows through the stellate cells. Consistent with this model, fluorescently labeled dextran, an in vivo marker of membrane water permeability, is trapped in the basal infoldings of the stellate cells after kinin diuretic peptide stimulation, confirming that these cells provide the major route for transepithelial water flux. The spatial segregation of these components of epithelial water transport may help to explain the unique success of the higher insects in regulating their internal environments.


Assuntos
Transporte Biológico/fisiologia , Drosophila melanogaster/fisiologia , Túbulos Renais/metabolismo , Água/metabolismo , Animais , Aquagliceroporinas/genética , Aquagliceroporinas/metabolismo , Aquaporinas/genética , Aquaporinas/metabolismo , Permeabilidade da Membrana Celular , Cloretos/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Túbulos Renais/citologia , Masculino , Túbulos de Malpighi/metabolismo , Modelos Animais , Oócitos/metabolismo , Osmorregulação , Xenopus
16.
Cell Prolif ; 53(3): e12763, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31925859

RESUMO

In recent years, although the development of clinical therapy for diabetic kidney disease (DKD) has made great progress, the progression of DKD still cannot be controlled. Therefore, further study of the pathogenesis of DKD and improvements in DKD treatment are crucial for prognosis. Traditional studies have shown that podocyte injury plays an important role in this process. Recently, it has been found that glomerulotubular balance and tubuloglomerular feedback (TGF) may be involved in the progression of DKD. Glomerulotubular balance is the specific gravity absorption of the glomerular ultrafiltrate by the proximal tubules, which absorbs only 65% to 70% of the ultrafiltrate. This ensures that the urine volume will not change much regardless of whether the glomerular filtration rate (GFR) increases or decreases. TGF is one of the significant mechanisms of renal blood flow and self-regulation of GFR, but how they participate in the development of DKD in the pathological state and the specific mechanism is not clear. Injury to tubular epithelial cells (TECs) is the key link in DKD. Additionally, injury to glomerular endothelial cells (GECs) plays a key role in the early occurrence and development of DKD. However, TECs and GECs are close to each other in anatomical position and can crosstalk with each other, which may affect the development of DKD. Therefore, the purpose of this review was to summarize the current knowledge on the crosstalk between TECs and GECs in the pathogenesis of DKD and to highlight specific clinical and potential therapeutic strategies.


Assuntos
Nefropatias Diabéticas/fisiopatologia , Glomérulos Renais/fisiopatologia , Túbulos Renais/fisiopatologia , Animais , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Progressão da Doença , Endotélio/citologia , Endotélio/metabolismo , Endotélio/patologia , Humanos , Glomérulos Renais/citologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Urotélio/citologia , Urotélio/metabolismo , Urotélio/patologia , Urotélio/fisiopatologia
17.
N Engl J Med ; 382(5): 416-426, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-31995687

RESUMO

BACKGROUND: Acute kidney injury is common, with a major effect on morbidity and health care utilization. Soluble urokinase plasminogen activator receptor (suPAR) is a signaling glycoprotein thought to be involved in the pathogenesis of kidney disease. We investigated whether a high level of suPAR predisposed patients to acute kidney injury in multiple clinical contexts, and we used experimental models to identify mechanisms by which suPAR acts and to assess it as a therapeutic target. METHODS: We measured plasma levels of suPAR preprocedurally in patients who underwent coronary angiography and patients who underwent cardiac surgery and at the time of admission to the intensive care unit in critically ill patients. We assessed the risk of acute kidney injury at 7 days as the primary outcome and acute kidney injury or death at 90 days as a secondary outcome, according to quartile of suPAR level. In experimental studies, we used a monoclonal antibody to urokinase plasminogen activator receptor (uPAR) as a therapeutic strategy to attenuate acute kidney injury in transgenic mice receiving contrast material. We also assessed cellular bioenergetics and generation of reactive oxygen species in human kidney proximal tubular (HK-2) cells that were exposed to recombinant suPAR. RESULTS: The suPAR level was assessed in 3827 patients who were undergoing coronary angiography, 250 who were undergoing cardiac surgery, and 692 who were critically ill. Acute kidney injury developed in 318 patients (8%) who had undergone coronary angiography. The highest suPAR quartile (vs. the lowest) had an adjusted odds ratio of 2.66 (95% confidence interval [CI], 1.77 to 3.99) for acute kidney injury and 2.29 (95% CI, 1.71 to 3.06) for acute kidney injury or death at 90 days. Findings were similar in the surgical and critically ill cohorts. The suPAR-overexpressing mice that were given contrast material had greater functional and histologic evidence of acute kidney injury than wild-type mice. The suPAR-treated HK-2 cells showed heightened energetic demand and mitochondrial superoxide generation. Pretreatment with a uPAR monoclonal antibody attenuated kidney injury in suPAR-overexpressing mice and normalized bioenergetic changes in HK-2 cells. CONCLUSIONS: High suPAR levels were associated with acute kidney injury in various clinical and experimental contexts. (Funded by the National Institutes of Health and others.).


Assuntos
Lesão Renal Aguda/sangue , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Angiografia Coronária/efeitos adversos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Lesão Renal Aguda/etiologia , Lesão Renal Aguda/prevenção & controle , Idoso , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Biomarcadores/sangue , Estado Terminal , Modelos Animais de Doenças , Feminino , Humanos , Unidades de Terapia Intensiva , Túbulos Renais/citologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Razão de Chances , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Complicações Pós-Operatórias/sangue , Complicações Pós-Operatórias/etiologia , Medição de Risco/métodos , Ativador de Plasminogênio Tipo Uroquinase/farmacologia
18.
Am J Physiol Renal Physiol ; 318(3): F576-F588, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961716

RESUMO

Nephrotoxicity is a known clinical complication of cisplatin that limits the use of this potent antitumor drug. Cyclic nucleotide phosphodiesterases (PDEs) play complex roles in physiology and pathology. PDE4, which is a member of the PDE family, has four subtypes (PDE4A-PDE4D), and PDE4B plays an important role in inflammation. Thus, in the present study, we investigated the effect of PDE4/PDE4B inhibition on renal function and inflammation in a cisplatin nephrotoxicity model. In mice, cisplatin enhanced mRNA and protein expression of PDE4B in renal tubules. After treatment with the PDE4 inhibitor cilomilast, cisplatin-induced renal dysfunction, renal tubular injury, tubular cell apoptosis, and inflammation were all improved. Next, after silencing PDE4B in vivo, we observed a protective effect against cisplatin nephrotoxicity similar to that of the PDE4 inhibitor. In vitro, cisplatin-induced renal tubular cell death was strikingly ameliorated by the PDE4 inhibitor and PDE4B knockdown along with the blockade of the inflammatory response. Considering the known roles of some cell survival pathways in antagonizing insults, we examined levels of PDE4-associated proteins sirtuin 1, phosphatidylinositol 3-kinase, and phosphorylated AKT in cisplatin-treated renal tubular cells with or without cilomilast treatment. Strikingly, cisplatin treatment downregulated the expression of the above proteins, and this effect was largely abolished by the PDE4 inhibitor. Together, these findings indicate the beneficial role of PDE4/PDE4B inhibition in treating cisplatin nephrotoxicity, possibly through antagonizing inflammation and restoring cell survival signaling pathways.


Assuntos
Lesão Renal Aguda/induzido quimicamente , Cisplatino/toxicidade , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Ácidos Cicloexanocarboxílicos/farmacologia , Inflamação/tratamento farmacológico , Nitrilos/farmacologia , Inibidores da Fosfodiesterase 4/farmacologia , Lesão Renal Aguda/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Ácidos Cicloexanocarboxílicos/uso terapêutico , Células Epiteliais/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Túbulos Renais/citologia , Masculino , Camundongos , Nitrilos/uso terapêutico , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo
19.
Cell Tissue Res ; 379(3): 549-559, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31773302

RESUMO

Diabetic nephropathy (DN) is one of the most devastating complications of diabetes. Connective tissue growth factor (CTGF) levels are up-regulated in patients with DN and in renal tubular epithelial cells (RTECs) exposed to high glucose (HG). The underlying epigenetic mechanism remains to be elucidated. In the present study, we investigate the role of myocardin-related transcription factor A (MRTF-A) in HG-induced CTGF transcription in RTECs. We report that in two different animal models of DN, one induced by streptozotocin (STZ) injection and the other induced by high-fat diet (HFD) feeding, MRTF-A deficiency attenuated CTGF induction in the kidneys. In cultured RTECs, MRTF-A knockdown similarly ameliorated CTGF induction by HG treatment. Upon CTGF induction, there was an increase in acetylated histone H3 (AcH3) and trimethylated H3K4 (H3K4Me3) on the CTGF promoter region accompanying a decrease in dimethylated H3K9 (H3K9Me2). MRTF-A ablation in vivo or depletion in vitro comparably dampened the accumulation of AcH3 and H3K4Me3 but restored H3K9Me2 on the CTGF promoter. Further analyses revealed that MRTF-A interacted with and recruited histone demethylase KDM3A to the CTGF promoter to activate transcription. KDM3A silencing equivalently weakened HG-induced CTGF induction in RTECs. In conclusion, MRTF-A contributes to HG-induced CTGF transcription via an epigenetic mechanism.


Assuntos
Fator de Crescimento do Tecido Conjuntivo/genética , Nefropatias Diabéticas/metabolismo , Transativadores/metabolismo , Animais , Fator de Crescimento do Tecido Conjuntivo/biossíntese , Nefropatias Diabéticas/genética , Modelos Animais de Doenças , Epigenômica/métodos , Células Epiteliais/metabolismo , Glucose/administração & dosagem , Glucose/metabolismo , Células HEK293 , Humanos , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transativadores/deficiência , Transativadores/genética , Transcrição Genética , Regulação para Cima
20.
Life Sci ; 241: 117108, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31786192

RESUMO

AIMS: Telmisartan (TLM), a highly selective angiotensin II type 1 receptor blocker (ARB) and partial PPAR-γ agonist, has versatile beneficial effects against oxidative stress, apoptosis, inflammatory responses and epithelial-mesenchymal transition (EMT). However, its underlying mechanism of inhibiting oxalate and calcium oxalate (CaOx) crystal-induced EMT by activating the PPAR-γ pathway remains unclear. MAIN METHODS: CCK-8 assays were used to evaluate the effects of TLM on cell viability. In addition, intracellular reactive oxygen species (ROS) levels were measured by the cell-permeable fluorogenic probe 2,7-dichlorofluorescein diacetate (DCFH-DA). Wound-healing and Transwell assays were used to evaluate the migration ability of HK2 cells exposed to oxalate. Moreover, immunofluorescence, immunohistochemistry and western blotting were used to examine the expression of E-cadherin, N-cadherin, vimentin and α-SMA and explore the underlying molecular mechanisms in HK2 cells and a stone-forming rat model. KEY FINDINGS: Our results showed that TLM treatment could protect HK2 cells from oxalate-induced cytotoxicity and oxidative stress injury. Additionally, TLM prevented EMT induction by oxalate and CaOx crystals via the PPAR-γ-AKT/STAT3/p38 MAPK-Snail pathway in vitro and in vivo. However, knockdown of PPAR-γ with small interfering RNA or the PPAR-γ-specific antagonist GW9662 abrogated these protective effects of TLM. SIGNIFICANCE: As a PPAR-γ agonist, TLM can ameliorate oxalate and CaOx crystal-induced EMT by exerting an antioxidant effect through the PPAR-γ-AKT/STAT3/p38 MAPK-Snail signaling pathway. Therefore, TLM can block EMT progression and could be a potential therapeutic agent for preventing and treating calcium oxalate urolithiasis formation and recurrence.


Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Oxalatos/toxicidade , PPAR gama/metabolismo , Telmisartan/farmacologia , Animais , Oxalato de Cálcio/toxicidade , Linhagem Celular , Transição Epitelial-Mesenquimal/fisiologia , Humanos , Túbulos Renais/citologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , PPAR gama/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Fator de Transcrição STAT3/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta1/toxicidade , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
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