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1.
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
2.
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
3.
Chem Biol Interact ; 316: 108926, 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31874164

RESUMO

Grape seed proanthocyanidin extract (GSPE) has been reported to exhibit a variety of protective effects, such as antioxidant, anti-atherosclerosis and other pharmacological effects. As a member of the complement system, complement component 3 (C3) deposition in the glomerulus is recognized as an important causative mediator of various kidney diseases. In this study, we aimed to identify the effect of GSPE on C3 in the chronic kidney fibrosis and evaluate the possible mechanism. We observed that administration of GSPE relieves inflammation and chronic renal fibrosis in mouse models of UUO. GSPE inhibited C3 secreted by macrophages to relieve renal interstitial inflammation. In vitro, we found that C3 stimulated HMGB1 translocation form nucleus to cytoplasm and promote the expression of pro-inflammatory cytokines including TGF-ß1 in primary renal tubular epithelial cells (PTEC), which could be inhibited by GSPE. Meanwhile, GSPE could also decreased HMGB1-induced EMT of PTEC through suppresses the HMGB1/TLR4/p65/TGF-ß1 pathway. In addition, the myofibroblast activation was inhibited by GSPE via TGF-ß1/Smad2/3 signaling pathways in normal rat kidney fibroblast (NRK-49F) cells. Overall, these observations provide that GSPE alleviates renal fibrosis by inhibiting the C3/HMGB1/TGF-ß1 pathway and could thus lead to find the potential therapy for the suppression of renal fibrosis.


Assuntos
Extrato de Sementes de Uva/farmacologia , Rim/efeitos dos fármacos , Proantocianidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Células Cultivadas , Complemento C3/metabolismo , Modelos Animais de Doenças , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fibrose , Extrato de Sementes de Uva/uso terapêutico , Proteína HMGB1/metabolismo , Rim/patologia , Nefropatias/metabolismo , Nefropatias/patologia , Nefropatias/prevenção & controle , Túbulos Renais/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proantocianidinas/uso terapêutico , Ratos , Fator de Crescimento Transformador beta1/metabolismo
4.
Int J Mol Sci ; 20(20)2019 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-31614781

RESUMO

BACKGROUND: Scattered tubular-like cells (STCs) proliferate and differentiate to support neighboring injured renal tubular cells during recovery from insults. Renal artery stenosis (RAS) induces renal ischemia and hypertension and leads to loss of kidney function, but whether RAS alters renal endogenous repair mechanisms, such as STCs, remains unknown. We hypothesize that RAS in swine modifies the messenger RNA (mRNA) profile of STCs, blunting their in vitro reparative capacity. METHODS: CD24+/CD133+ STCs were isolated from pig kidneys after 10-weeks of RAS or sham (n = 3 each) and their gene cargo analyzed using high-throughput mRNAseq. Expression profiles for upregulated and downregulated mRNAs in RAS-STCs were functionally interpreted by gene ontology analysis. STC activation was assessed by counting the total number of STCs in pig kidney sections using flow cytometry, whereas cell proliferation was assessed in vitro. RESULTS: Of all expressed genes, 1430 genes were upregulated and 315 downregulated in RAS- versus Normal-STCs. Expression of selected candidate genes followed the same fold change directions as the mRNAseq findings. Genes upregulated in RAS-STCs were involved in cell adhesion, extracellular matrix remodeling, and kidney development, whereas those downregulated in RAS-STCs are related to cell cycle and cytoskeleton. The percentage of STCs from dissociated kidney cells was higher in RAS versus Normal pigs, but their proliferation rate was blunted. CONCLUSIONS: Renal ischemia and hypertension in swine induce changes in the mRNA profile of STCs, associated with increased STC activation and impaired proliferation. These observations suggest that RAS may alter the reparative capacity of STCs.


Assuntos
Obstrução da Artéria Renal/genética , Transcriptoma , Animais , Células Cultivadas , Feminino , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Obstrução da Artéria Renal/metabolismo , Suínos
5.
Adv Exp Med Biol ; 1165: 101-116, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31399963

RESUMO

Kidney transplantation is a life-change measurement for the patients of end-stage renal disease (ESRD). However, the renal allograft cannot avoid initial acute kidney injury (AKI) and subsequent chronic allograft dysfunction (CAD), gradually develops fibrosis and eventually loses function. It is imperative to disclose the pathogenesis of AKI and CAD in order to facilitate interventions. We have studied the involvement of immunity, inflammation, and apoptosis in ischemia-reperfusion injury (IRI) and/or immunosuppressant induced AKI models, with associated chronic damage. Our research mainly focused on tubular epithelial cells (TECs) that are passive victims and also active participators in injury and mediate following repair or fibrosis. Targeting not only fibroblasts/myofibroblasts, but also TECs, might be a fundamental strategy to prevent and treat renal fibrosis. We have also evaluated the potential application of siRNA targeting caspase-3 and tissue protective erythropoietin derivatives, HBSP and CHBP, aiming to treat AKI and prevent CAD. Significant improvements have been obtained, but timely diagnosis and precise therapy of AKI and prevention of CAD progressing to ESRD are still very challenging. Modern technologies such as microarray and sequencing analysis have been used to identify biomarkers and potentially facilitate individual cell target treatment for transplant patients.


Assuntos
Lesão Renal Aguda , Transplante de Rim/efeitos adversos , Rim/patologia , Traumatismo por Reperfusão , Aloenxertos , Apoptose , Células Epiteliais/citologia , Fibrose , Humanos , Imunidade , Inflamação , Túbulos Renais/citologia
6.
Adv Exp Med Biol ; 1165: 233-252, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31399968

RESUMO

The renal tubules are the major component of the kidney and are vulnerable to a variety of injuries including ischemia, proteinuria, toxins, and metabolic disorders. It has long been believed that tubules are the victim of injury. In this review, we shift this concept to renal tubules as a driving force in the progression of kidney disease. In response to injury, tubular epithelial cells (TECs) can synthesize and secrete varieties of bioactive molecules that drive interstitial inflammation and fibrosis. Innate immune-sensing receptors on the TECs also aggravate immune responses. Necroinflammation, an auto-amplification loop between tubular cell death and interstitial inflammation, leads to the exacerbation of renal injury. Furthermore, TECs also play an active role in progressive renal injury via mechanisms associated with the conversion into collagen-producing fibroblast phenotype, cell cycle arrest at both G1/S and G2/M checkpoints, and metabolic disorder. Thus, a better understanding the mechanisms by which tubular injury drives AKI and CKD is necessary for the development of therapeutics to halt the progression of CKD.


Assuntos
Células Epiteliais/patologia , Nefropatias/fisiopatologia , Túbulos Renais/citologia , Pontos de Checagem do Ciclo Celular , Fibrose , Humanos , Imunidade Inata , Rim/patologia
7.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(4): 313-319, 2019 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-31167690

RESUMO

Objective To explore the mechanism of transdifferentiation of human renal tubular epithelial cells induced by high glucose based on reactive oxygen species (ROS)/NF-κB signaling pathway. Methods HK-2 normal human proximal tubular epithelial cells were randomly divided into blank control group, osmotic pressure control group, high glucose group and pyrrolidine dithiocarbamate (PDTC) group. Cell morphology was observed by phase contrast microscope. Cell viability was measured by MTT assay. Flow cytometry was used to detect intracellular ROS content. Malondialdehyde (MDA) content and superoxide dismutase activity were tested by ELISA. Western blot analysis was used to examine the protein levels of NF-κBp65, phosphorylated IκBα (p-IκBα) and IKKα, monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1). The expressions of NF-κBp65, epithelial cadherin (E-cadherin) and alpha smooth muscle actin (α-SMA) were assessed by immunocytochemistry. Results In high glucose group, the cells became spindle-shaped or irregular, with radial edges, enlarged intercellular space, decreased refractive index and irregular arrangement. At the same time, the cell activity decreased with the prolongation of treatment time, and the cell activity in PDTC group was higher than that in high glucose group. Compared with the blank control group, the content of ROS and MDA increased and the activity of SOD decreased in the high glucose group, while the content of ROS and MDA decreased and the activity of SOD increased in the PDTC group compared with the high glucose group. Compared with the blank group, the protein levels of NF-κBp65, p-IκBα, IKKα, MCP-1 and ICAM-1 increased in the high glucose group, while the protein levels above decreased in the PDTC group compared with the high glucose group. Compared with the blank group, the proportion of positive cells of NF-κBp65 and α-SMA increased and the proportion of positive cells of E-cadherin decreased in the high glucose group. Compared with the high glucose group, the proportion of positive cells of NF-κBp65 and α-SMA decreased and the proportion of positive cells of E-cadherin increased in the PDTC group. Conclusion High glucose can induce epithelial-mesenchymal transition in HK-2 cells. Activation of NF-κB signaling pathway mediated by ROS participates in the above process, which can be blocked by PDTC.


Assuntos
Transdiferenciação Celular , Células Epiteliais/citologia , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Linhagem Celular , Meios de Cultura , Glucose , Humanos , Túbulos Renais/citologia , Pirrolidinas , Tiocarbamatos
8.
Artif Cells Nanomed Biotechnol ; 47(1): 2205-2212, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31159592

RESUMO

Renal tubular damage caused by persistent high glucose environment has been found to contribute to diabetic nephropathy. This study explored the effects of lncRNA growth arrest-specific 5 (GAS5) on high glucose-stimulated human renal tubular epithelial HK-2 damage, as well as the possible internal molecular mechanism. Viability and apoptosis of HK-2 cells were assessed with the help of CCK-8 assay and Annexin V-FITC/PI staining, respectively. Cell transfection was used to change the expression of GAS5, miR-27a and BNIP3. We found that high glucose stimulation suppressed HK-2 cell viability but induced cell apoptosis. The expression of GAS5 was increased in HK-2 cells under high glucose environment. Silence of GAS5 mitigated the high glucose-caused HK-2 cell viability reduction and apoptosis. Overexpression of miR-27a reversed the effects of GAS5 on high glucose-stimulated HK-2 cells. Overexpression of BNIP3 aggravated the high glucose-caused HK-2 cell viability reduction, apoptosis and activation of JNK pathway. Knockdown of BNIP3 had opposite effects. In conclusion, this research further confirmed the pro-apoptotic roles of GAS5 in renal tubular epithelial cells under high glucose environment. Silence of GAS5 alleviated high glucose toxicity to human renal tubular epithelial HK-2 cells might be via down-regulating miR-27a and BNIP3, and then inactivating JNK pathway. Highlights HG suppresses HK-2 cell viability, but promotes cell apoptosis; HG enhances the expression of GAS5 in HK-2 cells; Silence of GAS5 alleviates the HG-caused HK-2 cell toxicity; miR-27a participates in the effects of GAS5 silencing on HG-stimulated HK-2 cells; BNIP3 is regulated by miR-27a and related to the HG toxicity to HK-2 cells.


Assuntos
Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Inativação Gênica , Glucose/toxicidade , Túbulos Renais/citologia , MicroRNAs/genética , RNA Longo não Codificante/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Longo não Codificante/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
9.
Cell Biochem Funct ; 37(4): 290-300, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31211440

RESUMO

Vascular endothelial growth factor C (VEGF-C) is an angiogenic and lymphangiogenic growth factor. Recent research has revealed the role for VEGF-C in regulating autophagy by interacting with a nontyrosine kinase receptor, neuropilin-2 (NRP-2). However, whether VEGF-C participates in regulating cell survival and autophagy in renal proximal tubular cells is unknown. To address this question, we employed a cell modal of serum deprivation to verify the role of VEGF-C and its receptor NRP-2 in regulating cell survival and autophagy in NRK52E cell lines. The results show that VEGF-C rescued the loss of cell viability induced by serum deprivation in a concentration-dependent manner. Furthermore, endogenous VEGF-C was knocked down in NRK52E cells by using specific small-interfering RNAs (siRNA), cells were more sensitive to serum deprivation-induced cell death. A similar increase in cell death rate was observed following NRP-2 depletion in serum-starved NRK52E cells. Autophagy activity in serum-starved NRK52E cells was confirmed by western blot analysis of microtubule-associated protein-1 chain 3 (LC3), immunofluorescence staining of endogenous LC3, and the formation of autophagosomes by electron microscopy. VEGF-C or NRP-2 depletion further increased LC3 expression induced by serum deprivation, suggesting that VEGF-C and NRP-2 were involved in controlling autophagy in NRK52E cells. We further performed autophagic flux experiments to identify that VEGF-C promotes the activation of autophagy in serum-starved NRK52E cells. Together, these results suggest for the first time that VEGF-C/NRP-2 axis promotes survival and autophagy in NRK52E cells under serum deprivation condition. SIGNIFICANCE OF THE STUDY: More researchers had focused on the regulation of autophagy in kidney disease. The effect of VEGF-C on cell death and autophagy in renal epithelial cells has not been examined. We first identified the VEGF-C as a regulator of cell survival and autophagy in NRK52E cell lines. And VEGF-C/NRP-2 may mediate autophagy by regulating the phosphorylation of 4EBP1 and P70S6K. VEGF-C treatment may be identified as a therapeutic target in renal injury repair due to its capacity to promote tubular cell survival in the future.


Assuntos
Autofagia , Células Epiteliais/citologia , Túbulos Renais/citologia , Neuropilina-2/metabolismo , Soro , Fator C de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Ratos , Transdução de Sinais
10.
Ren Fail ; 41(1): 455-466, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31163002

RESUMO

Purpose: To investigate whether Niban protein plays a role in renal interstitial fibrosis by regulating renal tubular epithelial cell apoptosis and explore the underlying mechanism. Methods: Unilateral ureteral obstruction (UUO) model was performed in C57B/6J mice, and divided into sham operation group and groups of days 3, days 7, and days 14. Niban expression was detected by immunohistochemistry and Western blot. TUNEL assays were used to detected apoptosis. Niban siRNA and overexpression Niban plasmid were transfected in HK-2 cells respectively to explore apoptosis related mechanisms of Niban during angiotensin II (AngII) - and endoplasmic reticulum (ER) stress-induced injury. Results: With the development of obstruction, Niban's expression decreased gradually while apoptosis increased. Silencing of Niban not only increased the AngII- and ER stress-induced apoptosis, but also promoted the expression of caspase 8, caspase 9, Bip, and Chop. Overexpression of Niban reduced AngII-induced apoptosis and the expression of caspase 8 and caspase 9. Conclusions: Niban protein is involved in apoptosis regulation in HK-2 cells, and most likely via caspase-dependent pathway.


Assuntos
Apoptose , Biomarcadores Tumorais/metabolismo , Nefropatias/patologia , Túbulos Renais/patologia , Proteínas de Neoplasias/metabolismo , Animais , Biomarcadores Tumorais/genética , Caspase 8/metabolismo , Caspase 9/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Células Epiteliais , Fibrose , Humanos , Nefropatias/etiologia , Túbulos Renais/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/genética , RNA Interferente Pequeno , Obstrução Ureteral/complicações
11.
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
12.
Mol Med Rep ; 19(6): 5115-5122, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059037

RESUMO

3,3'­Diindolylmethane (DIM) is a naturally derived indole compound found in the Brassica family of vegetables. DIM has several beneficial effects, including anti­cancer, anti­inflammatory and anti­angiogenic functions. However, the effects of DIM on acute kidney injury (AKI) stimulated by lipopolysaccharide (LPS) are poorly studied. In this present study, male BALB/c mouse models of AKI were established using intraperitoneal injections of 10 mg/kg LPS. DIM (40 mg/kg) was administered intraperitoneally 24 and 2 h before LPS exposure. The results indicated that DIM significantly mitigated histopathological changes in the kidneys and improved the levels of blood urea nitrogen and serum creatinine. DIM also suppressed the LPS­induced production of reactive oxygen species and cell apoptosis. Furthermore, DIM treatment significantly decreased the expression of NADPH oxidase 2 (NOX2) and NOX4 in LPS­treated mice. Therefore, DIM may exert its renoprotective actions by inhibiting NOX­mediated oxidative stress and the apoptosis of renal tubular epithelial cells.


Assuntos
Lesão Renal Aguda/prevenção & controle , Apoptose/efeitos dos fármacos , Indóis/farmacologia , Lipopolissacarídeos/toxicidade , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Lesão Renal Aguda/etiologia , Lesão Renal Aguda/patologia , Animais , Nitrogênio da Ureia Sanguínea , Creatinina/sangue , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Glutationa/metabolismo , Rim/patologia , Túbulos Renais/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NADPH Oxidases/genética , Espécies Reativas de Oxigênio/metabolismo
13.
PLoS One ; 14(4): e0214514, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30958843

RESUMO

The kidney is especially sensitive to diseases associated with overactivation of the complement system. While most of these diseases affect kidney glomeruli and the microvasculature, there is also evidence for tubulointerstitial deposition of complement factors. Complement inactivating factors on cell membranes comprise CD55, CD59 and CD46, which is also termed membrane cofactor protein (MCP). CD46 has been described as localized to glomeruli, but especially also to proximal tubular epithelial cells (RPTECs). However, human cell culture models to assess CD46 function on RPTECs are still missing. Therefore, we here performed gene editing of RPTEC/TERT1 cells generating a monoclonal CD46-/- cell line that did not show changes of the primary cell like characteristics. In addition, factor I and CD46-mediated cleavage of C4b into soluble C4c and membrane deposited C4d was clearly reduced in the knock-out cell line as compared to the maternal cells. Thus, human CD46-/- proximal tubular epithelial cells will be of interest to dissect the roles of the epithelium and the kidney in various complement activation mediated tubulointerstitial pathologies or in studying CD46 mediated uropathogenic internalization of bacteria. In addition, this gives proof-of-principle, that telomerized cells can be used in the generation of knock-out, knock-in or any kind of reporter cell lines without losing the primary cell characteristics of the maternal cells.


Assuntos
Sistemas CRISPR-Cas , Ativação do Complemento , Células Epiteliais/citologia , Técnicas de Inativação de Genes , Proteína Cofatora de Membrana/genética , Telomerase/metabolismo , Linhagem Celular , Complemento C4/química , Complemento C4b/química , Edição de Genes , Humanos , Túbulos Renais/citologia , Telômero/ultraestrutura , gama-Glutamiltransferase/metabolismo
14.
PLoS One ; 14(4): e0209056, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30964862

RESUMO

Voltage-sensing phosphatases (VSPs) are transmembrane proteins that couple changes in membrane potential to hydrolysis of inositol signaling lipids. VSPs catalyze the dephosphorylation of phosphatidylinositol phosphates (PIPs) that regulate diverse aspects of cell membrane physiology including cell division, growth and migration. VSPs are highly conserved among chordates, and their RNA transcripts have been detected in the adult and embryonic stages of frogs, fish, chickens, mice and humans. However, the subcellular localization and biological function of VSP remains unknown. Using reverse transcriptase-PCR (RT-PCR), we show that both Xenopus laevis VSPs (Xl-VSP1 and Xl-VSP2) mRNAs are expressed in early embryos, suggesting that both Xl-VSPs are involved in early tadpole development. To understand which embryonic tissues express Xl-VSP mRNA, we used in situ hybridization (ISH) and found Xl-VSP mRNA in both the brain and kidney of NF stage 32-36 embryos. By Western blot analysis with a VSP antibody, we show increasing levels of Xl-VSP protein in the developing embryo, and by immunohistochemistry (IHC), we demonstrate that Xl-VSP protein is specifically localized to the apical membrane of both embryonic and adult kidney tubules. We further characterized the catalytic activity of both Xl-VSP homologs and found that while Xl-VSP1 catalyzes 3- and 5-phosphate removal, Xl-VSP2 is a less efficient 3-phosphatase with different substrate specificity. Our results suggest that Xl-VSP1 and Xl-VSP2 serve different functional roles and that VSPs are an integral component of voltage-dependent PIP signaling pathways during vertebrate kidney tubule development and function.


Assuntos
Túbulos Renais/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Túbulos Renais/citologia , Túbulos Renais/embriologia , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/análise , Monoéster Fosfórico Hidrolases/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Proteínas de Xenopus/análise , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/genética
15.
Biochem Biophys Res Commun ; 512(2): 170-175, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30879772

RESUMO

This work was aimed to determine the effect of 17ß-estradiol (17ßE) on cell proliferation in human renal tubular epithelial cells (HRTEC) isolated from kidneys from pediatric subjects, as well as the role of estrogen receptors involved in the 17ßE proliferative response. Treatment with 17ßE (10 nmol/L, 24 h) significantly stimulated cell proliferation, measured by 5-bromo-2-deoxyuridine (BrdU) uptake, in HRTEC primary cultures and in tubular structures obtained by 3D cultured-HRTEC. Incubation of HRTEC with the G protein-coupled estrogen receptor 1 (GPER-1) agonist G-1 increased BrdU uptake. Incubation of HRTEC with 17ßE activated the classic estrogen receptor alpha (ERα) but not ERß. Treatment of HRTEC with the GPER-1 antagonist G-15, the ER inhibitor ICI182,780, or the ß-catenin inhibitor iCRT14, completely abrogated the increase in BrdU uptake induced by 17ßE. We also show that 17ßE stimulated ß-catenin protein expression and translocation to the nucleus of HRTEC, effects that were abrogated by G-15 and ICI 182,780. In conclusion, estradiol stimulates cell proliferation in HRTEC primary cultures through both ERα and GPER-1 estrogen receptors and involves ß-catenin activation.


Assuntos
Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Túbulos Renais/citologia , Receptores Estrogênicos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Proliferação de Células , Células Cultivadas , Criança , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Túbulos Renais/metabolismo
16.
Semin Nephrol ; 39(2): 215-226, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30827343

RESUMO

Drug attrition related to kidney toxicity remains a challenge in drug discovery and development. In vitro models established over the past 2 decades to supplement in vivo studies have improved the throughput capacity of toxicity evaluation, but usually suffer from low predictive value. To achieve a paradigm shift in the prediction of drug-induced kidney toxicity, two aspects are fundamental: increased physiological relevance of the kidney model, and use of appropriate toxicity end points. Recent studies have suggested that increasing the physiological relevance of kidney models can improve their sensitivity to drug-induced damage. Here, we discuss how advanced culture models, including modified cell lines, induced pluripotent stem cells, kidney organoid cultures, and microfluidic devices enhance in vivo similarity. To this end, culture models aim to increase the proximal tubule epithelial phenotype, reconstitute multiple tissue compartments and extracellular matrix, allow exposure to fluid shear stress, and enable interaction between multiple cell types. Applying computation-aided end points and novel biomarkers to advanced culture models will further improve sensitivity and clinical relevance of in vitro drug-induced toxicity prediction. Implemented at the right stage of drug discovery and development and coupled to high-content evaluation techniques, these models have the potential to reduce attrition and aid the selection of candidate drugs with an appropriate safety profile.


Assuntos
Lesão Renal Aguda/induzido quimicamente , Técnicas In Vitro , Túbulos Renais/citologia , Animais , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas , Dispositivos Lab-On-A-Chip , Organoides , Medição de Risco
17.
PLoS One ; 14(3): e0212991, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30845150

RESUMO

Tubulogenesis, the organization of epithelial cells into tubular structures, is an essential step during renal organogenesis as well as during the regeneration process of renal tubules after injury. In the present study, endothelial cell-derived factors that modulate tubule formation were examined using an in vitro human tubulogenesis system. When human renal proximal tubular epithelial cells (RPTECs) were cultured in gels, tubular structures with lumens were induced in the presence of hepatocyte growth factor (HGF). Aquaporin 1 was localized in the apical membrane of these tubular structures, suggesting that these structures are morphologically equivalent to renal tubules in vivo. HGF-induced tubule formation was significantly enhanced when co-cultured with human umbilical vein endothelial cells (HUVECs) or in the presence of HUVEC-conditioned medium (HUVEC-CM). Co-culture with HUVECs did not induce tubular structures in the absence of HGF. A phospho-receptor tyrosine kinase array revealed that HUVEC-CM markedly enhanced phosphorylation of Ret, glial cell-derived neurotrophic factor (GDNF) receptor, in HGF-induced tubular structures compared to those without HUVEC-CM. HUVECs produced GDNF, and RPTECs expressed both Ret and GDNF family receptor alpha1 (co-receptor). HGF-induced tubule formation was significantly enhanced by addition of GDNF. Interestingly, not only HGF but also GDNF significantly induced phosphorylation of the HGF receptor, Met. These data indicate that endothelial cell-derived GDNF potentiates the tubulogenic properties of HGF and may play a critical role in the epithelial-endothelial crosstalk during renal tubulogenesis as well as tubular regeneration after injury.


Assuntos
Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Túbulos Renais/crescimento & desenvolvimento , Técnicas de Cultura de Células , Meios de Cultivo Condicionados/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Comunicação Parácrina/fisiologia , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas Recombinantes/metabolismo
18.
Methods Mol Biol ; 1926: 87-102, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30742265

RESUMO

Nephrons, the functional units of the kidney, are derived from nephron progenitor cells (NPCs). Here, we describe methods to reconstruct nephron tissue via induction of NPCs from mouse and human pluripotent stem cells, which mimic multistep developmental signals in vivo. Induced NPCs differentiate into three-dimensional nephron structures, including glomerular podocytes and nephric tubules, which are useful for studying early stages of kidney specification and morphogenetic processes in the context of normal development or disease.


Assuntos
Túbulos Renais/citologia , Rim/citologia , Néfrons/citologia , Células-Tronco Pluripotentes/citologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos
19.
Methods Mol Biol ; 1926: 103-115, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30742266

RESUMO

Human pluripotent stem cells (hPSCs) have been well known for their ability to generate kidney cell types. We developed a protocol that utilizes a set of growth factors to give rise to kidney progenitors, which when differentiated further in a monolayer gives rise to podocyte precursors, mesangial cells, proximal and distal tubular epithelial cells, and collecting duct cells. This article describes in detail how to obtain each of these segment-specific kidney cell types from hPSCs. Once obtained as a homogenous population, these cells are invaluable for nephrotoxicity testing, for disease modeling, and in tissue engineering approaches such as 3D bioprinting and seeding on acellular matrices and scaffolds.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Rim/citologia , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Humanos , Túbulos Renais/citologia
20.
J Biol Inorg Chem ; 24(2): 235-246, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30701361

RESUMO

Fibronectin, an extracellular matrix (ECM) protein, has been thought to be involved in pathogenic mechanisms of kidney stone disease, especially calcium oxalate (CaOx) type. Nevertheless, its precise roles in modulation of CaOx crystal remained unclear. We thus performed a systematic evaluation of effects of fibronectin on CaOx monohydrate (COM) crystal (the major causative chemical crystal in kidney stone formation) in various stages of kidney stone pathogenesis, including crystallization, crystal growth, aggregation, adhesion onto renal tubular cells, and invasion through ECM in renal interstitium. The data showed that fibronectin significantly decreased crystallization, growth and adhesive capability of COM crystals in a dose-dependent manner. In contrast, COM crystal aggregation and invasion through ECM migration chamber were significantly enhanced by fibronectin in a dose-dependent fashion. Sequence analysis revealed three calcium-binding and six oxalate-binding domains in fibronectin. Immunofluorescence study confirmed binding of fibronectin to COM crystals. Additionally, calcium- and oxalate-affinity assays confirmed depletion of both calcium and oxalate ions after incubation with fibronectin. Moreover, calcium-saturated and oxalate-saturated forms of fibronectin markedly reduced the modulatory activities of fibronectin on COM crystallization, crystal growth, aggregation, and adhesion onto the cells. These data strongly indicate the dual functions of fibronectin, which serves as an inhibitor for COM crystallization, crystal growth and adhesion onto renal tubular cells, but on the other hand, acts as a promoter for COM crystal aggregation and invasion through ECM. Finally, its COM crystal modulatory activities are most likely mediated through binding with calcium and oxalate ions on the crystals and in their environment.


Assuntos
Oxalato de Cálcio/química , Matriz Extracelular/química , Fibronectinas/química , Túbulos Renais/química , Animais , Adesão Celular , Cristalização , Cães , Humanos , Túbulos Renais/citologia , Células Madin Darby de Rim Canino
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