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1.
Life Sci ; 258: 118153, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32738361

RESUMO

AIMS: Obesity-related glomerulopathy (ORG) is characterized by glomerulomegaly with or without focal and segmental glomerulosclerosis lesions. Isothiocyanate sulforaphane (SFN) can protect kidneys from ORG-related damages. In this study, we investigated the effects of SFN as a preventive therapy or intervention for ORG to reveal its mechanism of action. MAIN METHODS: We established a mouse obesity model with preventive SFN or N-acetylcysteine treatment for 2 months. Thereafter, we used nuclear factor erythroid 2-related factor 2-deficient (Nrf2-/-) and wild type mice in our ORG model with SFN treatment. Finally, we generated a corresponding mouse podocyte model in vitro. The body weight, wet weight of perirenal-and peritesticular fat, and urinary albumin/creatinine ratio were assessed. We used periodic acid-Schiff staining and electron microscopy to assess the function of the kidneys and podocytes. In addition, we evaluated the expression of Nrf2 and podocyte-specific proteins by western blotting. KEY FINDINGS: Treatment with SFN reduced body weight, organ-associated fat weight, and urinary albumin/creatinine ratio in both the preventive treatment and disease intervention regimens. SFN treated mice exhibited higher expression levels of podocyte-specific proteins and better podocyte function. However, treatment with SFN did not affect these parameters in obese Nrf2-/- mice. Light chain 3 of microtubule-associated protein 1-II and metallothionein had higher expression in the wild type than in the Nrf2-/- mice. SIGNIFICANCE: Treatment with SFN limited ORG-induced damage by enhancing podocyte autophagy via Nrf2.


Assuntos
Isotiocianatos/uso terapêutico , Nefropatias/tratamento farmacológico , Nefropatias/etiologia , Fator 2 Relacionado a NF-E2/metabolismo , Obesidade/complicações , Substâncias Protetoras/uso terapêutico , Animais , Autofagia/efeitos dos fármacos , Células Cultivadas , Nefropatias/metabolismo , Glomérulos Renais/citologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Podócitos/citologia , Podócitos/efeitos dos fármacos , Podócitos/metabolismo
2.
Ecotoxicol Environ Saf ; 204: 111061, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32750588

RESUMO

The use of hexavalent chromium (Cr(VI)) in many industrial processes has resulted in serious environmental pollution problems. Cr(VI) causes organ toxicity in animals after ingestion or inhalation. However, the exact mechanism by which Cr(VI) produces kidney damage remains elusive. Herein, we investigated whether Cr(VI)-induced kidney damage is related to the disorder of mitochondrial dynamics. In this study, 28 male rats were divided into four groups and intraperitoneally injected with 0, 2, 4, and 6 mg/kg body weight potassium dichromate for 5 weeks. Experiment included analysis of renal histopathology and ultrastructure, determination of biochemical indicators, and measurement of related protein content. The results showed that Cr(VI) induced kidney injury through promotion of oxidative stress, apoptosis, and disorder of mitochondrial dynamics in a dose-dependent manner. The protein levels of the silent information regulator two ortholog 1 (Sirt1), peroxisome proliferation-activated receptor-g coactivator-1a (PGC-1a), and autophagy-related proteins were significantly decreased after Cr(VI) exposure. These findings suggest that Cr(VI) leads to the disorder of mitochondrial dynamics by inhibiting the Sirt1/PGC-1a pathway, which leads to renal apoptosis and autophagy in rats.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cromo/toxicidade , Rim/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Rim/metabolismo , Rim/ultraestrutura , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Dicromato de Potássio/toxicidade , Ratos , Ratos Wistar
3.
Am J Physiol Renal Physiol ; 319(2): F284-F291, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32686524

RESUMO

Podocyte dysfunction contributes to proteinuric chronic kidney disease. A number of key proteins are essential for podocyte function, including nephrin, podocin, CD2-associated protein (CD2AP), synaptopodin, and α-actinin-4 (ACTN4). Although most of these proteins were first identified through genetic studies associated with human kidney disease, subsequent studies have identified phosphorylation of these proteins as an important posttranslational event that regulates their function. In this review, a brief overview of the function of these key podocyte proteins is provided. Second, the role of phosphorylation in regulating the function of these proteins is described. Third, the association between these phosphorylation pathways and kidney disease is reviewed. Finally, challenges and future directions in studying phosphorylation are discussed. Better characterization of these phosphorylation pathways and others yet to be discovered holds promise for translating this knowledge into new therapies for patients with proteinuric chronic kidney disease.


Assuntos
Glomérulos Renais/metabolismo , Proteínas de Membrana/metabolismo , Fosforilação/fisiologia , Podócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas do Citoesqueleto/metabolismo , Humanos , Nefropatias/metabolismo
4.
Tohoku J Exp Med ; 251(2): 87-90, 2020 06.
Artigo em Inglês | MEDLINE | ID: covidwho-593619

RESUMO

In light of the recent pandemic, favipiravir (Avigan®), a purine nucleic acid analog and antiviral agent approved for use in influenza in Japan, is being studied for the treatment of coronavirus disease 2019 (COVID-19). Increase in blood uric acid level is a frequent side effect of favipiravir. Here, we discussed the mechanism of blood uric acid elevation during favipiravir treatment. Favipiravir is metabolized to an inactive metabolite M1 by aldehyde oxidase and xanthine oxidase, and excreted into urine. In the kidney, uric acid handling is regulated by the balance of reabsorption and tubular secretion in the proximal tubules. Favipiravir and M1 act as moderate inhibitors of organic anion transporter 1 and 3 (OAT1 and OAT3), which are involved in uric acid excretion in the kidney. In addition, M1 enhances uric acid reuptake via urate transporter 1 (URAT1) in the renal proximal tubules. Thus, favipiravir is thought to decrease uric acid excretion into urine, resulting in elevation of uric acid levels in blood. Elevated uric acid levels were returned to normal after discontinuation of favipiravir, and favipiravir is not used for long periods of time for the treatment of viral infection. Thus, the effect on blood uric acid levels was subclinical in most studies. Nevertheless, the adverse effect of favipiravir might be clinically important in patients with a history of gout, hyperuricemia, kidney function impairment (in which blood concentration of M1 increases), and where there is concomitant use of other drugs affecting blood uric acid elevation.


Assuntos
Amidas/efeitos adversos , Antivirais/efeitos adversos , Infecções por Coronavirus/tratamento farmacológico , Hiperuricemia/induzido quimicamente , Pneumonia Viral/tratamento farmacológico , Pirazinas/efeitos adversos , Ácido Úrico/sangue , Aldeído Oxidase/metabolismo , Amidas/farmacocinética , Amidas/urina , Antivirais/farmacocinética , Biotransformação , Interações Medicamentosas , Humanos , Hiperuricemia/fisiopatologia , Rim/metabolismo , Nefropatias/metabolismo , Estrutura Molecular , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Pandemias , Pirazinas/farmacocinética , Pirazinas/urina , Xantina Oxidase/metabolismo
5.
Life Sci ; 256: 117901, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32504759

RESUMO

AIMS: Cyclophosphamide (CTX) is an effective anti-tumor and immunosuppressive agent, but it induces nephrotoxicity in clinical applications. The present study aimed to evaluate the protective effect of pyrroloquinoline quinone (PQQ) on CTX-induced nephrotoxicity. MAIN METHODS: We injected male ICR mice with CTX (80 mg/kg/day), and determined nephrotoxicity indices, MDA and antioxidant defenses, inflammatory cytokines, and the levels of main proteins in the Nrf2-HO-1 and NLRP3 signaling pathways. KEY FINDINGS: PQQ has significantly decreased the serum levels of creatinine and urea compared to Model group. When treated with PQQ, MDA, IL-1ß, IL-6, and TNF-α levels have decreased, and SOD, GSH-Px, and CAT activity have increased in the kidney tissues of CTX-induced mice. PQQ activated the Nrf2-mediated signaling pathway, as indicated by the increased expression of Nrf2, HO-1, GCLM, and NQO1. Moreover, PQQ inhibited the NLRP3 inflammatory pathway, as indicated by the reduced expression of NLRP3, ASC, and Caspase-1. SIGNIFICANCE: Our results suggest that PQQ protects against CTX-induced nephrotoxicity, probably by activating the Nrf2-mediated antioxidant pathway and inhibiting the NLRP3 inflammatory pathway.


Assuntos
Ciclofosfamida/efeitos adversos , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Cofator PQQ/uso terapêutico , Transdução de Sinais , Animais , Antioxidantes/metabolismo , Nitrogênio da Ureia Sanguínea , Peso Corporal/efeitos dos fármacos , Creatinina/metabolismo , Citocinas/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Rim/efeitos dos fármacos , Rim/enzimologia , Rim/patologia , Masculino , Malondialdeído/metabolismo , Camundongos Endogâmicos ICR , Modelos Biológicos , Tamanho do Órgão/efeitos dos fármacos , Cofator PQQ/química , Cofator PQQ/farmacologia
6.
Tohoku J Exp Med ; 251(2): 87-90, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32536670

RESUMO

In light of the recent pandemic, favipiravir (Avigan®), a purine nucleic acid analog and antiviral agent approved for use in influenza in Japan, is being studied for the treatment of coronavirus disease 2019 (COVID-19). Increase in blood uric acid level is a frequent side effect of favipiravir. Here, we discussed the mechanism of blood uric acid elevation during favipiravir treatment. Favipiravir is metabolized to an inactive metabolite M1 by aldehyde oxidase and xanthine oxidase, and excreted into urine. In the kidney, uric acid handling is regulated by the balance of reabsorption and tubular secretion in the proximal tubules. Favipiravir and M1 act as moderate inhibitors of organic anion transporter 1 and 3 (OAT1 and OAT3), which are involved in uric acid excretion in the kidney. In addition, M1 enhances uric acid reuptake via urate transporter 1 (URAT1) in the renal proximal tubules. Thus, favipiravir is thought to decrease uric acid excretion into urine, resulting in elevation of uric acid levels in blood. Elevated uric acid levels were returned to normal after discontinuation of favipiravir, and favipiravir is not used for long periods of time for the treatment of viral infection. Thus, the effect on blood uric acid levels was subclinical in most studies. Nevertheless, the adverse effect of favipiravir might be clinically important in patients with a history of gout, hyperuricemia, kidney function impairment (in which blood concentration of M1 increases), and where there is concomitant use of other drugs affecting blood uric acid elevation.


Assuntos
Amidas/efeitos adversos , Antivirais/efeitos adversos , Infecções por Coronavirus/tratamento farmacológico , Hiperuricemia/induzido quimicamente , Pneumonia Viral/tratamento farmacológico , Pirazinas/efeitos adversos , Ácido Úrico/sangue , Aldeído Oxidase/metabolismo , Amidas/farmacocinética , Amidas/urina , Antivirais/farmacocinética , Biotransformação , Interações Medicamentosas , Humanos , Hiperuricemia/fisiopatologia , Rim/metabolismo , Nefropatias/metabolismo , Estrutura Molecular , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Pandemias , Pirazinas/farmacocinética , Pirazinas/urina , Xantina Oxidase/metabolismo
7.
Am J Physiol Renal Physiol ; 319(2): F155-F161, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32538149

RESUMO

Systemic lupus erythematosus (SLE) is characterized by hypertension that results from chronic renal inflammation and dysautonomia in the form of dampened vagal tone. In health, the vagus nerve regulates inflammatory processes through mechanisms like the cholinergic anti-inflammatory pathway; so in the case of SLE, reduced efferent vagus nerve activity may indirectly affect renal inflammation and therefore hypertension. In this study, we sought to investigate the impact of disrupting vagal neurotransmission on renal inflammation and hypertension in the setting of chronic inflammatory disease. Female SLE (NZBWF1) and control (NZW) mice were subjected to a right unilateral cervical vagotomy or sham surgery and 3 wk later were implanted with indwelling catheters to measure blood pressure. Indices of splenic and renal inflammation, as well as renal injury, were assessed. Unilateral vagotomy blunted SLE-induced increases in mean arterial pressure, albumin excretion rate, and glomerulosclerosis. This protection was associated with reduced splenic T cells and attenuated SLE-induced increases in renal proinflammatory mediators. In summary, these data indicate that unilateral vagotomy reduces renal inflammation and reduces blood pressure in SLE mice. The vagus nerves have myriad functions, and perhaps other neuroimmune interactions compensate for the ligation of one nerve.


Assuntos
Pressão Sanguínea/fisiologia , Inflamação/metabolismo , Rim/lesões , Rim/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Albuminúria/fisiopatologia , Animais , Modelos Animais de Doenças , Hipertensão/complicações , Hipertensão/fisiopatologia , Nefropatias/metabolismo , Lúpus Eritematoso Sistêmico/complicações , Camundongos Endogâmicos , Nefrite/metabolismo , Nefrite/fisiopatologia
8.
Am J Physiol Renal Physiol ; 319(1): F93-F105, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32475133

RESUMO

The long noncoding RNA nuclear enriched abundant transcript 1 (NEAT1) has been reported to promote liver fibrosis progression. However, its molecular mechanism in renal fibrosis was not elucidated. In the present study, an in vitro model of renal fibrosis was established with HK-2 and HKC-8 cells treated with transforming growth factor-ß1. C57BL/6 mice were used for the in vivo model with unilateral ureteral obstruction. Our results indicated that NEAT1 and collagen type I levels were significantly upregulated, whereas miR-129 was obviously downregulated, in the progression of renal fibrosis. Meanwhile, NEAT1 knockdown or miR-129 overexpression inhibited collagen type I deposition, the epithelial-mesenchymal transition process, and the inflammation response to suppress renal fibrosis. NEAT1 directly targeted miR-129, and miR-129 directly bound to collagen type I. Downregulation of miR-129 reversed inhibition of renal fibrosis induced by NEAT1 silencing, and upregulation of collagen type I also reversed inhibition of renal fibrosis caused by miR-129 overexpression. NEAT1 knockdown alleviated renal fibrosis in mice subjected to unilateral ureteral obstruction. In conclusion, NEAT1 sponged miR-129 to modulate the epithelial-mesenchymal transition process and inflammation response of renal fibrosis by regulation of collagen type I. Our study indicates a novel role in the regulation of renal fibrosis and provides a new potential treatment target for renal fibrosis.


Assuntos
Colágeno Tipo I/metabolismo , Nefropatias/metabolismo , Rim/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Nitrogênio da Ureia Sanguínea , Linhagem Celular , Creatinina/sangue , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibrose/metabolismo , Fibrose/patologia , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Nefropatias/patologia , Camundongos , MicroRNAs/genética , RNA Longo não Codificante/genética , Fator de Crescimento Transformador beta1/farmacologia
9.
Proc Natl Acad Sci U S A ; 117(27): 15862-15873, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32561647

RESUMO

Albuminuria is an independent risk factor for the progression to end-stage kidney failure, cardiovascular morbidity, and premature death. As such, discovering signaling pathways that modulate albuminuria is desirable. Here, we studied the transcriptomes of podocytes, key cells in the prevention of albuminuria, under diabetic conditions. We found that Neuropeptide Y (NPY) was significantly down-regulated in insulin-resistant vs. insulin-sensitive mouse podocytes and in human glomeruli of patients with early and late-stage diabetic nephropathy, as well as other nondiabetic glomerular diseases. This contrasts with the increased plasma and urinary levels of NPY that are observed in such conditions. Studying NPY-knockout mice, we found that NPY deficiency in vivo surprisingly reduced the level of albuminuria and podocyte injury in models of both diabetic and nondiabetic kidney disease. In vitro, podocyte NPY signaling occurred via the NPY2 receptor (NPY2R), stimulating PI3K, MAPK, and NFAT activation. Additional unbiased proteomic analysis revealed that glomerular NPY-NPY2R signaling predicted nephrotoxicity, modulated RNA processing, and inhibited cell migration. Furthermore, pharmacologically inhibiting the NPY2R in vivo significantly reduced albuminuria in adriamycin-treated glomerulosclerotic mice. Our findings suggest a pathogenic role of excessive NPY-NPY2R signaling in the glomerulus and that inhibiting NPY-NPY2R signaling in albuminuric kidney disease has therapeutic potential.


Assuntos
Albuminúria/metabolismo , Nefropatias/metabolismo , Neuropeptídeo Y/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Transdução de Sinais/fisiologia , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Benzazepinas/farmacologia , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas , Modelos Animais de Doenças , Regulação para Baixo , Doxorrubicina/farmacologia , Humanos , Insulina/metabolismo , Nefropatias/patologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neuropeptídeo Y/farmacologia , Neuropeptídeo Y/urina , Podócitos/metabolismo , Proteômica , Receptores de Neuropeptídeo Y/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
10.
Ren Fail ; 42(1): 513-522, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32441195

RESUMO

Objective: To investigate the way that miR-136 regulated spleen tyrosine kinase (SYK) and transforming growth factor-ß1 (TGF-ß1)/Smad3 signaling pathways on renal fibrosis.Methods: 100 male SD (Sprague-Dawley) rats were randomly divided into diabetic nephropathy (DN) group, normal control (NC) group, miR-136 mimics group, and control group. The renal fibrosis model of diabetic rats was established by streptozotocin (STZ) method. NRK-52E cells were transfected into six groups: HG group, HG + miR-136 group, HG + miR-NC group, miR-136 + SYK group, miR-136 + NC group, and control group. Histopathological examination, the expressions of miR-136 and SYK mRNA, the expression of mTOR, blood glucose, urine protein, body weight, creatinine level, blood urea nitrogen (BUN), and KW/BW were detected in each group. Transfection efficiency, the targeted binding, and regulation between miR-136 and SYK, as well as the expression level of related inflammatory factors, the expression levels of SYK, E-Cad (E-cadherin), Vimentin, Collagen I, α-smooth muscle actin (α-SMA), and vascular endothelial growth factor A (VEGFA) were detected.Results: It was shown that the expression level of miR-136 in DN group significantly decreased. The blood glucose and urine protein concentrations in the DN group and miR-136 mimics group significantly increased and the body weight was decreased, but the blood glucose concentration in the miR-136 mimics group increased with time. The prolongation of the decline significantly decreased, and the growth rate of urinary protein reduced. Creatinine, BUN, and the kidney weight to body weight ratio (KW/BW) in DN group increased significantly. Cell culture results showed that SYK was a target gene of miR-136 and miR-136/SYK-mediated renal fibrosis by activating TGF-ß1/Smad3 signal.Conclusion: SYK activates TGF-ß1/Smad3 signaling, while miR-136 inhibits TGF-ß1/Smad3 signaling mediating tubular epithelial cell fibrosis by down-regulating SYK.


Assuntos
Nefropatias/metabolismo , Nefropatias/patologia , MicroRNAs/genética , Transdução de Sinais , Quinase Syk/metabolismo , Animais , Linhagem Celular , Regulação para Baixo , Fibrose/genética , Fibrose/metabolismo , Nefropatias/genética , Masculino , Ratos , Ratos Sprague-Dawley , Proteína Smad3/metabolismo , Quinase Syk/genética , Fator de Crescimento Transformador beta1/metabolismo
11.
Metabolism ; 108: 154258, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32376130

RESUMO

RATIONALE: Tubulointerstitial fibrosis, which is closely related to functional injury of the kidney, can be observed in advanced stages of diabetic nephropathy (DN). Mammalian serine/threonine-protein kinase 4 (MST1), a core component of the Hippo pathway that is involved in cellular proliferation and differentiation, plays a crucial role in the pathogenesis of multiple metabolic diseases, kidney diseases and cancer. METHODS: In type 1 and type 2 diabetic animals, as well as in human proximal tubular epithelial cells (HK-2), activation of MST1 was analyzed by immunohistochemistry and western blotting. In db/db mice, MST1 protein was knocked down or overexpressed by shRNA, and renal function, fibrosis, and downstream signaling were then investigated. RNA silencing and overexpression were performed by using an MST1 or YAP knockdown/expression lentivirus to investigate the regulation of MST1-mediated YAP/TEAD signaling pathways in the fibrosis process in HK-2 cells. Luciferase and coimmunoprecipitation (co-IP) assays were used to identify whether YAP directly regulated TEAD activation by forming a YAP-TEAD heterodimer, which ultimately leads to tubulointerstitial fibrosis. RESULTS: MST1 activation was significantly decreased in type 1 and type 2 diabetic nephropathy. Notably, the downregulation of MST1 activation was also observed in HK-2 cells in a glucose- and time-dependent manner. In vivo, downregulation of MST1 was sufficient to promote renal dysfunction and fibrosis in db/m mice, whereas overexpression of MST1 ameliorated diabetic nephropathy-induced renal fibrosis. Further mechanistic study demonstrated that activated YAP induced by MST1 inhibition directly upregulated TEAD activation by binding to TEAD and forming a YAP-TEAD heterodimer, resulting in the promotion of epithelial-mesenchymal transition (EMT) and fibrosis in renal tubular epithelial. CONCLUSIONS: MST1 activation represents a potential therapeutic strategy to treat or prevent the progression of diabetic nephropathy-induced renal fibrosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diabetes Mellitus/metabolismo , Nefropatias Diabéticas/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Fibrose/metabolismo , Nefropatias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Regulação para Baixo/fisiologia , Regulação da Expressão Gênica/fisiologia , Glucose/metabolismo , Túbulos Renais/metabolismo , Camundongos , Ratos , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Regulação para Cima/fisiologia
12.
PLoS One ; 15(5): e0233109, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32437461

RESUMO

Normalisation to standard reference gene(s) is essential for quantitative real-time polymerase chain reaction (RT-qPCR) to obtain reproducible and comparable results of a gene of interest (GOI) between subjects and under varying experimental conditions. There is limited evidence to support selection of the commonly used reference genes in rat ischaemic and toxicological kidney models. Employing these models, we determined the most stable reference genes by comparing 4 standard methods (NormFinder, qBase+, BestKeeper and comparative ΔCq) and developed a new 3-way linear mixed-effects model for evaluation of reference gene stability. This new technique utilises the intra-class correlation coefficient as the stability measure for multiple continuous and categorical covariates when determining the optimum normalisation factor. The model also determines confidence intervals for each candidate normalisation gene to facilitate selection and allow sample size calculation for designing experiments to identify reference genes. Of the 10 candidate reference genes tested, the geometric mean of polyadenylate-binding nuclear protein 1 (PABPN1) and beta-actin (ACTB) was the most stable reference combination. In contrast, commonly used ribosomal 18S and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were the most unstable. We compared the use of PABPN1×ACTB and 2 commonly used genes 18S and GAPDH on the expression of 4 genes of interest know to vary after renal injury and expressed by different kidney cell types (KIM-1, HIF1α, TGFß1 and PECAM1). The less stable reference genes gave varying patterns of GOI expression in contrast to the use of the least unstable reference PABPN1×ACTB combination; this improved detection of differences in gene expression between experimental groups. Reduced within-group variation of the now more accurately normalised GOI may allow for reduced experimental group size particularly for comparison between various models. This objective selection of stable reference genes increased the reliability of comparisons within and between experimental groups.


Assuntos
Regulação da Expressão Gênica , Isquemia/metabolismo , Nefropatias/metabolismo , Rim/irrigação sanguínea , Rim/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Actinas/biossíntese , Animais , Isquemia/patologia , Rim/patologia , Nefropatias/patologia , Proteína I de Ligação a Poli(A)/biossíntese , RNA Ribossômico 18S/biossíntese , Ratos , Padrões de Referência
13.
Cardiovasc Pathol ; 48: 107218, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32388447

RESUMO

Cardiac amyloid A (AA) amyloidosis is rare. We present the case of a 72-year-old woman with obstructive hypertrophic cardiomyopathy (HCM) and biopsy-proven renal AA amyloidosis whose dyspnea and exercise intolerance had worsened over the previous year. Her AA amyloidosis was suspected to be secondary to chronic diverticulitis for which she had undergone hemicolectomy and sigmoidectomy 3 years prior. Echocardiographic findings were consistent with worsening left ventricular outflow tract obstruction at rest. Cardiac magnetic resonance imaging revealed patchy areas of midwall late gadolinium enhancement. Right ventricular endomyocardial biopsy did not reveal amyloid deposition, and cardiac technetium-99m pyrophosphate scintigraphy did not suggest transthyretin amyloidosis. The patient underwent septal myectomy with resection of an accessory papillary muscle. Pathological examination of the myectomy specimen was consistent with HCM. In addition, there was a thick layer of diffuse endocardial and vascular amyloid deposition that was identified as AA type by laser-microdissection with liquid chromatography-coupled tandem-mass spectrometry. This case report highlights the presence of 2 distinct disease processes occurring simultaneously and the importance of tissue diagnosis of AA amyloidosis, a condition that is not commonly associated with HCM.


Assuntos
Amiloidose/complicações , Cardiomiopatia Hipertrófica/complicações , Insuficiência Cardíaca/etiologia , Nefropatias/complicações , Miocárdio/patologia , Obstrução do Fluxo Ventricular Externo/etiologia , Idoso , Amiloidose/metabolismo , Amiloidose/patologia , Amiloidose/fisiopatologia , Procedimentos Cirúrgicos Cardíacos , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/patologia , Cardiomiopatia Hipertrófica/fisiopatologia , Feminino , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Nefropatias/metabolismo , Nefropatias/patologia , Miocárdio/metabolismo , Proteína Amiloide A Sérica/metabolismo , Resultado do Tratamento , Função Ventricular Esquerda , Obstrução do Fluxo Ventricular Externo/metabolismo , Obstrução do Fluxo Ventricular Externo/patologia , Obstrução do Fluxo Ventricular Externo/fisiopatologia
14.
Phytomedicine ; 72: 153232, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32460034

RESUMO

BACKGROUND: In chronic kidney disease, although fibrosis prevention is beneficial, few interventions are available that specifically target fibrogenesis. Poricoic acid A (PAA) isolated from Poria cocos exhibits anti-fibrotic effects in the kidney, however the underlying mechanisms remain obscure. PURPOSE: We isolated PAA and investigated its effects and the underlying mechanisms in renal fibrosis. STUDY DESIGN: Unilateral ureteral obstruction (UUO) and 5/6 nephrectomy (Nx) animal models and TGF-ß1-induced renal fibroblasts (NRK-49F) were used to investigate the anti-fibrotic activity of PAA and its underlying mechanisms. METHODS: Western blots, qRT-PCR, immunofluorescence staining, co-immunoprecipitation and molecular docking methods were used. Knock-down and knock-in of adenosine monophosphate-activated protein kinase (AMPK) in the UUO model and cultured NRK-49F cells were employed to verify the mechanisms of action of PAA. RESULTS: PAA improved renal function and alleviated fibrosis by stimulating AMPK and inhibiting Smad3 specifically in Nx and UUO models. Reduced AMPK activity was associated with Smad3 induction, fibroblast activation, and the accumulation and aberrant remodelling of extracellular matrix (ECM) in human renal puncture samples and cultured NRK-49F cells. PAA stimulated AMPK activity and decreased fibrosis in a dose-dependent manner, thus showing that AMPK was essential for PAA to exert its anti-fibrotic effects. AMPK deficiency reduced the anti-fibrotic effects of PAA, while AMPK overexpression enhanced its effect. CONCLUSION: PAA activated AMPK and further inhibited Smad3 specifically to suppress fibrosis by preventing aberrant ECM accumulation and remodelling and facilitating the deactivation of fibroblasts.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Matriz Extracelular/efeitos dos fármacos , Nefropatias/tratamento farmacológico , Rim/patologia , Triterpenos/farmacologia , Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/genética , Animais , Estudos de Casos e Controles , Linhagem Celular , Relação Dose-Resposta a Droga , Matriz Extracelular/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Ratos Sprague-Dawley , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Triterpenos/química , Obstrução Ureteral/tratamento farmacológico , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia
16.
Nat Commun ; 11(1): 1943, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32327648

RESUMO

Kidney fibrosis is a highly deleterious process and a final manifestation of chronic kidney disease. Alpha-(α)-synuclein (SNCA) is an actin-binding neuronal protein with various functions within the brain; however, its role in other tissues is unknown. Here, we describe the expression of SNCA in renal epithelial cells and demonstrate its decrease in renal tubules of murine and human fibrotic kidneys, as well as its downregulation in renal proximal tubular epithelial cells (RPTECs) after TGF-ß1 treatment. shRNA-mediated knockdown of SNCA in RPTECs results in de novo expression of vimentin and α-SMA, while SNCA overexpression represses TGF-ß1-induced mesenchymal markers. Conditional gene silencing of SNCA in RPTECs leads to an exacerbated tubulointerstitial fibrosis (TIF) in two unrelated in vivo fibrotic models, which is associated with an increased activation of MAPK-p38 and PI3K-Akt pathways. Our study provides an evidence that disruption of SNCA signaling in RPTECs contributes to the pathogenesis of renal TIF by facilitating partial epithelial-to-mesenchymal transition and extracellular matrix accumulation.


Assuntos
Nefropatias/patologia , Rim/patologia , alfa-Sinucleína/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibrose , Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Rim/metabolismo , Nefropatias/genética , Nefropatias/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Obstrução Ureteral/genética , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , Vimentina/genética , Vimentina/metabolismo , alfa-Sinucleína/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
17.
Am J Physiol Renal Physiol ; 318(5): F1160-F1166, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32174141

RESUMO

Renal fibrosis is a major contributor to the development and progression of chronic kidney disease. A low-protein diet can reduce the progression of chronic kidney disease and reduce the development of renal fibrosis, although the mechanism is not well understood. Urea reabsorption into the inner medulla is regulated by inner medullary urea transporter (UT)-A1 and UT-A3. Inhibition or knockout of UT-A1/A3 will reduce interstitial urea accumulation, which may be beneficial in reducing renal fibrosis. To test this hypothesis, the effect of unilateral ureteral obstruction (UUO) was compared in wild-type (WT) and UT-A1/A3 knockout mice. UUO causes increased extracellular matrix associated with increases in transforming growth factor-ß, vimentin, and α-smooth muscle actin (α-SMA). In WT mice, UUO increased the abundance of three markers of fibrosis: transforming growth factor-ß, vimentin, and α-SMA. In contrast, in UT-A1/A3 knockout mice, the increase following UUO was significantly reduced. Consistent with the Western blot results, immunohistochemical staining showed that the levels of vimentin and α-SMA were increased in WT mice with UUO and that the increase was reduced in UT-A1/A3 knockout mice with UUO. Masson's trichrome staining showed increased collagen in WT mice with UUO, which was reduced in UT-A1/A3 knockout mice with UUO. We conclude that reduced UT activity reduces the severity of renal fibrosis following UUO.


Assuntos
Nefropatias/metabolismo , Rim/patologia , Proteínas de Membrana Transportadoras/deficiência , Obstrução Ureteral/complicações , Actinas/metabolismo , Animais , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Fibrose , Rim/metabolismo , Nefropatias/etiologia , Nefropatias/patologia , Nefropatias/prevenção & controle , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Índice de Gravidade de Doença , Fator de Crescimento Transformador beta/metabolismo , Obstrução Ureteral/genética , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , Vimentina/metabolismo
18.
Am J Physiol Renal Physiol ; 318(5): F1122-F1135, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32174138

RESUMO

Emerging evidence has demonstrated that (pro)renin receptor (PRR)-mediated activation of intrarenal renin-angiotensin system (RAS) plays an essential role in renal handling of Na+ and water balance and blood pressure. The present study tested the possibility that the intrarenal RAS served as a molecular target for the protective action of ELABELA (ELA), a novel endogenous ligand of apelin receptor, in the distal nephron. By RNAscope and immunofluorescence, mRNA and protein expression of endogenous ELA was consistently localized to the collecting duct (CD). Apelin was also found in the medullary CDs as assessed by immunofluorescence. In cultured CD-derived M1 cells, exogenous ELA induced parallel decreases of full-length PRR (fPRR), soluble PRR (sPRR), and prorenin/renin protein expression as assessed by immunoblotting and medium sPRR and prorenin/renin levels by ELISA, all of which were reversed by 8-bromoadenosine 3',5'-cyclic monophosphate. Conversely, deletion of PRR in the CD or nephron in mice elevated Apela and Apln mRNA levels as well as urinary ELA and apelin excretion, supporting the antagonistic relationship between the two systems. Administration of exogenous ELA-32 infusion (1.5 mg·kg-1·day-1, minipump) to high salt (HS)-loaded Dahl salt-sensitive (SS) rats significantly lowered mean arterial pressure, systolic blood pressure, diastolic blood pressure, and albuminuria, accompanied with a reduction of urinary sPRR, angiotensin II, and prorenin/renin excretion. HS upregulated renal medullary protein expression of fPRR, sPRR, prorenin, and renin in Dahl SS rats, all of which were significantly blunted by exogenous ELA-32 infusion. Additionally, HS-induced upregulation of inflammatory cytokines (IL-1ß, IL-2, IL-6, IL-17A, IFN-γ, VCAM-1, ICAM-1, and MCP-1), fibrosis markers (TGF-ß1, FN, Col1A1, PAI-1, and TIMP-1), and kidney injury markers (NGAL, Kim-1, albuminuria, and urinary NGAL excretion) were markedly blocked by exogenous ELA infusion. Together, these results support the antagonistic interaction between ELA and intrarenal RAS in the distal nephron that appears to exert a major impact on blood pressure regulation.


Assuntos
Pressão Sanguínea , Hipertensão/metabolismo , Nefropatias/metabolismo , Rim/metabolismo , Hormônios Peptídicos/metabolismo , Sistema Renina-Angiotensina , Animais , Apelina/genética , Apelina/metabolismo , Receptores de Apelina/genética , Receptores de Apelina/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Hipertensão/tratamento farmacológico , Hipertensão/fisiopatologia , Rim/efeitos dos fármacos , Rim/patologia , Rim/fisiopatologia , Nefropatias/patologia , Nefropatias/fisiopatologia , Nefropatias/prevenção & controle , Masculino , Camundongos Knockout , Hormônios Peptídicos/administração & dosagem , Hormônios Peptídicos/genética , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Ratos Endogâmicos Dahl , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Transdução de Sinais
19.
Phytomedicine ; 69: 153185, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32120244

RESUMO

BACKGROUND: Dihydroquercetin (DHQ) is an antifibrotic agent. However, whether DHQ can prevent renal fibrosis remains unknown. PURPOSE: This study aimed to investigate the effects of DHQ on tubulointerstitial fibrosis and its underlying mechanisms in unilateral ureteral obstruction (UUO) mice in vivo and NRK-49F cells in vitro. METHODS: In vivo, UUO mice received vehicle or DHQ treatment. In vitro, NRK-49F cells were pretreated with DHQ and exposed to transforming growth factor-ß1 (TGF-ß1). Changes in fibroblast activation, collagen synthesis, oxidative stress, and related signaling pathways were assessed by immunohistochemical staining, Western blot analysis, real-time reverse transcription-PCR, and fluorescence microscopy. RESULTS: UUO induced tubular atrophy, inflammation, fibroblast differentiation into myofibroblast, and collagen deposition, whereas DHQ ameliorated these effects. UUO also resulted in decreased levels of nuclear factor-erythroid-2-related factor 2 (Nrf2), catalase, and heme oxygenase-1, but increased H2O2 and malondialdehyde levels. DHQ treatment corrected these changes. In vitro, the intracellular Nrf2 level of NRK-49F exposed to TGF-ß1 decreased. However, DHQ rescued intracellular Nrf2 level and promoted nuclear translocation of Nrf2. DHQ scavenged TGF-ß1-induced accumulation of reactive oxygen species, inhibited TGF-ß1-induced Smad3 phosphorylation, and prevented TGF-ß1-induced fibroblast activation and collagen synthesis in NRK-49F. Nrf2 knockdown could suppress the DHQ-mediated inhibitory effects on oxidative stress, Smad3 phosphorylation, fibroblast activation, and collagen deposition. Furthermore, DHQ ameliorated established renal fibrosis in UUO mice. CONCLUSIONS: DHQ posed remarkable preventive and therapeutic effects on UUO-induced renal fibrosis and suppressed fibroblast activation by reducing oxidative stress and Smad3 phosphorylation via Nrf2 signaling. This study provided a mechanistic basis for the clinical application of DHQ in renal fibrosis treatment.


Assuntos
Nefropatias/tratamento farmacológico , Fator 2 Relacionado a NF-E2/metabolismo , Substâncias Protetoras/farmacologia , Quercetina/análogos & derivados , Transdução de Sinais/efeitos dos fármacos , Animais , Fibrose , Peróxido de Hidrogênio/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Quercetina/química , Quercetina/farmacologia , Ratos , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia
20.
Biol Pharm Bull ; 43(3): 533-539, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32115512

RESUMO

Renal interstitial fibrosis (RIF) is a common pathological characteristic associated with end-stage renal disease. However, treatment strategies for RIF are still very limited. In this study, we reported that kaempferol, a classic flavonoid, exhibited strong and widely inhibitory effect on the expression of fibrosis related genes in transforming growth factor beta 1 (TGF-ß1) treated NRK-52E cells. Further studies revealed that kaempferol inhibited TGF-ß1 induced epithelial-mesenchymal transition (EMT) process of NRK-52E cells and improved renal function deterioration and RIF in unilateral ureteral obstruction (UUO) rats. After exploring the underlying mechanisms, we found that kaempferol was able to activate the BMP-7-Smad1/5 pathway, rather than the TGF-ß1-Smad2/3 pathway. To further validate these results, DMH1 and BMP-7 knockdown were utilized at the cellular level and the results showed that both methods were able to antagonize the effects of kaempferol on the EMT process of NRK-52E cells induced by TGF-ß1. In UUO rats, inhibition of BMP-7 signaling by DMH1 also reversed the effects of kaempferol on renal function decline and RIF. Taken together, our findings demonstrated that kaempferol could be a good candidate for renal fibrosis treatment.


Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Quempferóis/farmacologia , Nefropatias/metabolismo , Proteínas Smad Reguladas por Receptor/metabolismo , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Animais , Linhagem Celular , Colágeno/metabolismo , Células Epiteliais , Fibrose , Rim/efeitos dos fármacos , Rim/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo
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