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2.
Medicine (Baltimore) ; 99(39): e22341, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32991448

RESUMO

RATIONALE: The Goodpasture syndrome is an extremely rare disease, with renal and pulmonary manifestations, and is mediated by anti-glomerular basement membrane (anti-GBM) antibodies. Renal pathological changes are mainly characterized by glomerular crescent formation and linear immunofluorescent staining for immunoglobulin G on the GBM. There are few reports on the atypical course of the syndrome involving serum-negative anti-GBM antibodies. Therefore, we present a case of Goodpasture syndrome that presented with nephrotic-range proteinuria and was seronegative for anti-GBM antibodies. PATIENT CONCERNS: A 38-year-old Chinese man presented with a lung lesion that was discovered by physical examination a month prior to presentation. The chief concern was occasional hemoptysis without fever, cough, chest pain, and edema. DIAGNOSES: Laboratory testing revealed that the urinary protein level and urine erythrocyte count were 7.4 g/24 hours and 144/high-power field (HPF), respectively. Serological testing for anti-GBM antibodies was negative. Chest computed tomography revealed multiple exudative lesions in both lungs, indicating alveolar infiltration and hemorrhage. Electronic bronchoscopy and pathological examination of the alveolar lavage fluid indicated no abnormalities. However, kidney biopsy suggested cellular crescent formation and segmental necrosis of the globuli, with linear IgG and complement C3 deposition on the GBM. These findings were consistent with the diagnosis of anti-GBM antibody nephritis. INTERVENTIONS: The patient underwent 7 sessions of double filtration plasmapheresis. He was also administered with intravenous methylprednisolone and cyclophosphamide. After renal function stabilization, he was discharged under an immunosuppressive regimen comprising of glucocorticoids and cyclophosphamides. OUTCOMES: Three months later, follow-up examination revealed that the 24-hour urine protein had increased to 13 g. Furthermore, the urine erythrocyte count was 243/HPF. After a 6-month follow-up, the patient achieved partial remission, with a proteinuria level of 3.9 g/24 hours and a urine erythrocyte count of 187/HPF. LESSONS: This extremely rare case of Goodpasture syndrome manifested with seronegativity for anti-GBM antibodies and nephrotic-range proteinuria. Our findings emphasize the importance of renal biopsy for the clinical diagnosis of atypical cases. Furthermore, because renal involvement achieved only partial remission despite therapy, early detection and active treatment of the Goodpasture syndrome is necessary to improve the prognosis of patients.


Assuntos
Doença Antimembrana Basal Glomerular/complicações , Doença Antimembrana Basal Glomerular/imunologia , Autoanticorpos/sangue , Proteinúria/etiologia , Administração Intravenosa , Adulto , Assistência ao Convalescente , Doença Antimembrana Basal Glomerular/diagnóstico , Doença Antimembrana Basal Glomerular/terapia , Grupo com Ancestrais do Continente Asiático/etnologia , Complemento C3/metabolismo , Ciclofosfamida/administração & dosagem , Ciclofosfamida/uso terapêutico , Quimioterapia Combinada , Glucocorticoides/administração & dosagem , Glucocorticoides/uso terapêutico , Hemoptise/diagnóstico , Hemorragia/etiologia , Hemorragia/patologia , Humanos , Imunoglobulina G/metabolismo , Imunossupressores/administração & dosagem , Imunossupressores/uso terapêutico , Rim/patologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Pulmão/diagnóstico por imagem , Pulmão/patologia , Masculino , Metilprednisolona/administração & dosagem , Metilprednisolona/uso terapêutico , Nefrite/diagnóstico , Nefrite/imunologia , Plasmaferese/métodos , Tomografia Computadorizada por Raios X/métodos , Resultado do Tratamento
3.
Life Sci ; 259: 118281, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32798554

RESUMO

AIMS: Intrauterine growth restriction (IUGR) can increase the risk of hypertension and kidney disease at adulthood due to fetal programming. In our previous study, we found that supplementation with low concentration of ouabain during pregnancy could restore glomerulus numbers at birth, rescuing kidney development. However, the metabolic pattern of kidney in IUGR offspring and the effect of ouabain have not been evaluated. MAIN METHODS: In this study, based on GC-MS and LC-MS platforms, we used the protein restriction rat model to explore the molecular mechanisms of kidney damage induced by IUGR and the protective effect of ouabain. KEY FINDINGS: The results showed that malnutrition could induce IUGR in rat offspring at the 20th gestational day but ouabain treatment could partially reverse the body and kidney weight loss. Ouabain treatment could upregulate arginine, N-acetylornithine and carbamoyl phosphate as well as adenine nucleotide and guanine nucleotide downregulated by low-protein diet. Moreover, six metabolites were identified to be significantly correlated with fetal kidney weight, with 3 metabolites involved in arginine metabolism (arginine, N-acetylornithine, urea) and UDP-glucuronate correlated positively, while lysine and anthranilate correlated negatively. SIGNIFICANCE: The results suggested that the underlying mechanism of ouabain against renal maldevelopment involved the metabolic regulation, particularly the arginine metabolism, which played an important role in the development of fetal kidney.


Assuntos
Retardo do Crescimento Fetal/metabolismo , Rim/metabolismo , Ouabaína/farmacologia , Animais , Arginina/metabolismo , Dieta com Restrição de Proteínas , Feminino , Desenvolvimento Fetal/efeitos dos fármacos , Retardo do Crescimento Fetal/fisiopatologia , Peso Fetal/efeitos dos fármacos , Hipertensão/fisiopatologia , Rim/efeitos dos fármacos , Nefropatias/metabolismo , Glomérulos Renais/metabolismo , Masculino , Metabolômica , Ouabaína/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Ratos , Ratos Sprague-Dawley
4.
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
5.
J Vis Exp ; (160)2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32628162

RESUMO

Glomerular cell death is a pathological feature of myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV). Extracellular deoxyribonucleic acid (ecDNA) is released during different forms of cell death including apoptosis, necrosis, necroptosis, neutrophil extracellular traps (NETs) and pyroptosis. Measurement of this cell death is time consuming with several different biomarkers required to identify the different biochemical forms of cell death. Measurement of ecDNA is generally conducted in serum and urine as a surrogate for renal damage, not in the actual target organ where the pathological injury occurs. The current difficulty in investigating ecDNA in the kidney is the lack of methods for formalin fixed paraffin embedded tissue (FFPE) both experimentally and in archived human kidney biopsies. This protocol provides a summary of the steps required to stain for ecDNA in FFPE tissue (both human and murine), quench autofluorescence and measure the ecDNA in the resulting images using a machine learning tool from the publicly available open source ImageJ plugin trainable Weka segmentation. Trainable Weka segmentation is applied to ecDNA within the glomeruli where the program learns to classify ecDNA. This classifier is applied to subsequent acquired kidney images, reducing the need for manual annotations of each individual image. The adaptability of the trainable Weka segmentation is demonstrated further in kidney tissue from experimental murine anti-MPO glomerulonephritis (GN), to identify NETs and ecMPO, common pathological contributors to anti-MPO GN. This method provides objective analysis of ecDNA in kidney tissue that demonstrates clearly the efficacy in which the trainable Weka segmentation program can distinguish ecDNA between healthy normal kidney tissue and diseased kidney tissue. This protocol can easily be adapted to identify ecDNA, NETs and ecMPO in other organs.


Assuntos
DNA/análise , Espaço Extracelular/metabolismo , Glomerulonefrite/genética , Aprendizado de Máquina Supervisionado , Animais , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/sangue , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/patologia , Biópsia , Armadilhas Extracelulares/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Peroxidase/metabolismo
6.
Am J Physiol Renal Physiol ; 319(2): F312-F322, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32628542

RESUMO

The ultrafiltrate flow over the major processes and cell body generates fluid flow shear stress (FFSS) on podocytes. Hyperfiltration-associated increase in FFSS can lead to podocyte injury and detachment. Previously, we showed that FFSS-induced upregulation of the cyclooxygenase 2 (COX2)-PGE2-prostaglandin E receptor 2 (EP2) axis in podocytes activates Akt-glycogen synthase kinase-3ß-ß-catenin and MAPK/ERK signaling in response to FFSS. Integrative MultiOmics Pathway Resolution (IMPRes) is a new bioinformatic tool that enables simultaneous time-series analysis of more than two groups to identify pathways and molecular connections. In the present study, we used previously characterized COX2 [prostaglandin-endoperoxide synthase 2 (Ptgs2)], EP2 (Ptger2), and ß1-catenin (Ctnnb1) as "seed genes" from an array data set of four groups analyzed over a time course. The 3 seed genes shared 7 pathways and 50 genes of 14 pathways and 89 genes identified by IMPRes. A composite of signaling pathways highlighted the temporal molecular connections during mechanotransduction signaling in FFSS-treated podocytes. We investigated the "proteoglycans in cancer" and "galactose metabolism" pathways predicted by IMPRes. A custom-designed PCR array validated 60.7% of the genes predicted by IMPRes analysis, including genes for the above-named pathways. Further validation using Western blot analysis showed increased expression of phosho-Erbb2, phospho-mammalian target of rapamycin (mTOR), CD44, and hexokinase II (Hk2); decreased total Erbb2, galactose mutarotase (Galm), and ß-1,4-galactosyltransferase 1 (B4galt1); and unchanged total mTOR and AKT3. These findings corroborate our previously reported results. This study demonstrates the potential of the IMPRes method to identify novel pathways. Identifying the "proteoglycans in cancer" and "galactose metabolism" pathways has generated a lead to study the significance of FFSS-induced glycocalyx remodeling and possible detachment of podocytes from the glomerular matrix.


Assuntos
Podócitos/metabolismo , Proteoglicanas/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Estresse Mecânico , Ativação Transcricional/fisiologia , Ciclo-Oxigenase 2/metabolismo , Glomérulos Renais/metabolismo , Mecanotransdução Celular/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima
7.
Am J Physiol Renal Physiol ; 319(2): F335-F344, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32657157

RESUMO

Human immunodeficiency virus (HIV) infection of kidney cells can lead to HIV-associated nephropathy (HIVAN) and aggravate the progression of other chronic kidney diseases. Thus, a better understanding of the mechanisms of HIV-induced kidney cell injury is needed for effective therapy against HIV-induced kidney disease progression. We have previously shown that the acetylation and activation of key inflammatory regulators, NF-κB p65 and STAT3, were increased in HIVAN kidneys. Here, we demonstrate the key role of sirtuin 1 (SIRT1) deacetylase in the regulation of NF-κB and STAT3 activity in HIVAN. We found that SIRT1 expression was reduced in the glomeruli of human and mouse HIVAN kidneys and that HIV-1 gene expression was associated with reduced SIRT1 expression and increased acetylation of NF-κB p65 and STAT3 in cultured podocytes. Interestingly, SIRT1 overexpression, in turn, reduced the expression of negative regulatory factor in podocytes stably expressing HIV-1 proviral genes, which was associated with inactivation of NF-κB p65 and a reduction in HIV-1 long terminal repeat promoter activity. In vivo, the administration of the small-molecule SIRT1 agonist BF175 or inducible overexpression of SIRT1 specifically in podocytes markedly attenuated albuminuria, kidney lesions, and expression of inflammatory markers in Tg26 mice. Finally, we showed that the reduction in SIRT1 expression by HIV-1 is in part mediated through miR-34a expression. Together, our data provide a new mechanism of SIRT1 regulation and its downstream effects in HIV-1-infected kidney cells and indicate that SIRT1/miR-34a are potential drug targets to treat HIV-related kidney disease.


Assuntos
Nefropatia Associada a AIDS/virologia , Insuficiência Renal Crônica/metabolismo , Sirtuína 1/metabolismo , Nefropatia Associada a AIDS/complicações , Nefropatia Associada a AIDS/metabolismo , Animais , Humanos , Rim/metabolismo , Glomérulos Renais/metabolismo , Glomérulos Renais/virologia , Camundongos , Podócitos/metabolismo , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/virologia , Fator de Transcrição RelA/metabolismo
8.
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
9.
Clin Sci (Lond) ; 134(12): 1433-1448, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32478392

RESUMO

Recent identification of an RNA-binding protein (HuR) that regulates mRNA turnover and translation of numerous transcripts via binding to an ARE in their 3'-UTR involved in inflammation and is abnormally elevated in varied kidney diseases offers a novel target for the treatment of renal inflammation and subsequent fibrosis. Thus, we hypothesized that treatment with a selective inhibition of HuR function with a small molecule, KH-3, would down-regulate HuR-targeted proinflammatory transcripts thereby improving glomerulosclerosis in experimental nephritis, where glomerular cellular HuR is elevated. Three experimental groups included normal and diseased rats treated with or without KH-3. Disease was induced by the monoclonal anti-Thy 1.1 antibody. KH-3 was given via daily intraperitoneal injection from day 1 after disease induction to day 5 at the dose of 50 mg/kg BW/day. At day 6, diseased animals treated with KH-3 showed significant reduction in glomerular HuR levels, proteinuria, podocyte injury determined by ameliorated podocyte loss and podocin expression, glomerular staining for periodic acid-Schiff positive extracellular matrix proteins, fibronectin and collagen IV and mRNA and protein levels of profibrotic markers, compared with untreated disease rats. KH-3 treatment also reduced disease-induced increases in renal TGFß1 and PAI-1 transcripts. Additionally, a marked increase in renal NF-κB-p65, Nox4, and glomerular macrophage cell infiltration observed in disease control group was largely reversed by KH-3 treatment. These results strongly support our hypothesis that down-regulation of HuR function with KH-3 has therapeutic potential for reversing glomerulosclerosis by reducing abundance of pro-inflammatory transcripts and related inflammation.


Assuntos
Proteína Semelhante a ELAV 1/antagonistas & inibidores , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Nefrite/metabolismo , Nefrite/patologia , Animais , Biomarcadores/metabolismo , Peso Corporal , Polaridade Celular , Colágeno/genética , Colágeno/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrose , Humanos , Inflamação/patologia , Testes de Função Renal , Glomérulos Renais/fisiopatologia , Macrófagos/metabolismo , Masculino , Monócitos/metabolismo , NADPH Oxidase 4/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Antígenos Thy-1 , Fator de Transcrição RelA/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
10.
Sci Rep ; 10(1): 9419, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32523052

RESUMO

Dominant and recessive mutations in podocalyxin (PODXL) are associated with human kidney disease. Interestingly, some PODXL mutations manifest as anuria while others are associated with proteinuric kidney disease. PODXL heterozygosity is associated with adult-onset kidney disease and podocalyxin shedding into the urine is a common biomarker of a variety nephrotic syndromes. It is unknown, however, how various lesions in PODXL contribute to these disparate disease pathologies. Here we generated two mouse stains: one that deletes Podxl in developmentally mature podocytes (Podxl∆Pod) and a second that is heterozygous for podocalyxin in all tissues (Podxl+/-). We used histologic and ultrastructural analyses, as well as clinical chemistry assays to evaluate kidney development and function in these strains. In contrast to null knockout mice (Podxl-/-), which die shortly after birth from anuria and hypertension, Podxl∆Pod mice develop an acute congenital nephrotic syndrome characterized by focal segmental glomerulosclerosis (FSGS) and proteinuria. Podxl+/- mice, in contrast, have a normal lifespan, and fail to develop kidney disease under normal conditions. Intriguingly, although wild-type C57Bl/6 mice are resistant to puromycin aminonucleoside (PA)-induced nephrosis (PAN), Podxl+/- mice are highly sensitive and PA induces severe proteinuria and collapsing FSGS. In summary, we find that the developmental timepoint at which podocalyxin is ablated (immature vs. mature podocytes) has a profound effect on the urinary phenotype due to its critical roles in both the formation and the maintenance of podocyte ultrastructure. In addition, Podxl∆Pod and Podxl+/- mice offer powerful new mouse models to evaluate early biomarkers of proteinuric kidney disease and to test novel therapeutics.


Assuntos
Nefropatias/metabolismo , Podócitos/metabolismo , Sialoglicoproteínas/metabolismo , Animais , Feminino , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Heterozigoto , Humanos , Nefropatias/patologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Síndrome Nefrótica/metabolismo , Síndrome Nefrótica/patologia , Fenótipo , Podócitos/patologia , Proteinúria/metabolismo , Proteinúria/patologia , Puromicina Aminonucleosídeo/metabolismo
11.
Nat Commun ; 11(1): 2777, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488001

RESUMO

Currently, there are no treatments for Alport syndrome, which is the second most commonly inherited kidney disease. Here we report the development of an exon-skipping therapy using an antisense-oligonucleotide (ASO) for severe male X-linked Alport syndrome (XLAS). We targeted truncating variants in exon 21 of the COL4A5 gene and conducted a type IV collagen α3/α4/α5 chain triple helix formation assay, and in vitro and in vivo treatment efficacy evaluation. We show that exon skipping enabled trimer formation, leading to remarkable clinical and pathological improvements including expression of the α5 chain on glomerular and the tubular basement membrane. In addition, the survival period was clearly prolonged in the ASO treated mice group. This data suggests that exon skipping may represent a promising therapeutic approach for treating severe male XLAS cases.


Assuntos
Colágeno Tipo IV/metabolismo , Éxons/fisiologia , Nefrite Hereditária/metabolismo , Nefrite Hereditária/terapia , Animais , Colágeno Tipo IV/química , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Células HEK293 , Humanos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Camundongos , Modelos Moleculares , Nefrite Hereditária/genética , Nefrite Hereditária/patologia , Insuficiência Renal Crônica
12.
Life Sci ; 257: 118010, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32598932

RESUMO

Podocyte injury is an early event and core in the development of focal segmental glomerular sclerosis (FSGS) that induces poor prognosis. Epithelial-mesenchymal transition (EMT) as a response of podocyte to injury leads to podocyte depletion and proteinuria. The abnormally reactivated NOTCH pathway may be involved in podocyte EMT. Baicalin, as a natural flavonoid compound, had significant inhibitory activity on tissue fibrosis and tumor cell invasion. However, its potential role and molecular mechanisms to injured podocyte in FSGS are little known. Here we found that baicalin could inhibit podocyte EMT markers expression and cell migration induced by TGF-ß1, accompanied by the up-regulated expression of slit diaphragm (SD) proteins and cell-cell adhesion molecule. Further investigation revealed that EMT inhibition of baicalin on injured podocyte is mainly mediated by the reduction of notch1 activation and its downstream Snail expression. Using the adriamycin-induced FSGS model, we determined that baicalin suppresses the Notch1-Snail axis activation in podocytes, relieves glomerulus structural disruption and dysfunction, and reduces proteinuria. Altogether, these findings suggest that baicalin is a novel renoprotective agent against podocyte EMT in FSGS and indicate its underlying mechanism that involves in negative regulation of the Notch1-Snail axis.


Assuntos
Flavonoides/farmacologia , Glomerulosclerose Segmentar e Focal/tratamento farmacológico , Proteinúria/tratamento farmacológico , Animais , Biomarcadores/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Doxorrubicina/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Flavonoides/metabolismo , Glomerulosclerose Segmentar e Focal/metabolismo , Glomérulos Renais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Podócitos/metabolismo , Proteinúria/metabolismo , Receptor Notch1/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
13.
Am J Physiol Renal Physiol ; 319(2): F245-F255, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32567348

RESUMO

Ca2+ is an important second messenger that translates extracellular stimuli into intracellular responses. Although there has been significant progress in understanding Ca2+ dynamics in organs such as the brain, the nature of Ca2+ signals in the kidney is still poorly understood. Here, we show that by using a genetically expressed highly sensitive reporter (GCaMP6s), it is possible to perform imaging of Ca2+ signals at high resolution in the mouse kidney in vivo. Moreover, by applying machine learning-based automated analysis using a Ca2+-independent signal, quantitative data can be extracted in an unbiased manner. By projecting the resulting data onto the structure of the kidney, we show that different tubular segments display highly distinct spatiotemporal patterns of Ca2+ signals. Furthermore, we provide evidence that Ca2+ activity in the proximal tubule decreases with increasing distance from the glomerulus. Finally, we demonstrate that substantial changes in intracellular Ca2+ can be detected in proximal tubules in a cisplatin model of acute kidney injury, which can be linked to alterations in cell structure and transport function. In summary, we describe a powerful new tool to investigate how single cell behavior is integrated with whole organ structure and function and how it is altered in disease states relevant to humans.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Glomérulos Renais/metabolismo , Túbulos Renais Proximais/metabolismo , Lesão Renal Aguda/metabolismo , Animais , Humanos , Rim/anatomia & histologia , Rim/metabolismo , Túbulos Renais Proximais/anatomia & histologia , Camundongos
14.
Lab Invest ; 100(9): 1169-1183, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32472097

RESUMO

Type 1 diabetes is a T-cell mediated autoimmune disease characterized by pancreatic beta cells destruction. Angiotensin-converting enzyme 2 (ACE2), a component of renin-angiotensin system (RAS) has been identified in pancreas from type 2 diabetic mice and its overexpression prevents beta cell dysfunction. We studied the effect of ACE2 deletion on pancreatic and renal function in the nonobese diabetic mice, a model that mimics type 1 diabetes. ACE2-deficient NOD mice and the respective controls were generated. Pancreas function and immunohistochemistry studies were performed. Renal function and RAS gene expression were also analyzed. Renal proximal tubular cells were obtained from these animals to dissect the effect of ACE2 deficiency in these cells. In NOD mice, ACE2 deletion significantly worsened glucose homeostasis, decreased islet insulin content, increased beta cell oxidative stress, and RIPK1-positive islets as compared with control mice. Angiotensin-converting enzyme and angiotensin II type 1 receptor (AT1R) were also increased in ACE2-deficient mice. In kidneys of 30-day diabetic mice, ACE2 deletion decreased podocyte number within the glomeruli, and altered renal RAS gene expression in tubules. ACE2 deletion influenced the expression of fibrosis-related genes in isolated primary renal proximal tubular cells before diabetes onset in NOD mice. Our findings suggest that ACE2 deletion may have a deleterious impact on beta cell and renal function, by promoting oxidative stress and increasing necroptosis mediators. In addition, this effect is accompanied by RAS alterations in both pancreas and renal proximal tubular cells, indicating that ACE2 may exert a renopancreatic protective effect on type 1 diabetes, which is activated before diabetes starts.


Assuntos
Diabetes Mellitus Tipo 1/genética , Rim/metabolismo , Pâncreas/metabolismo , Peptidil Dipeptidase A/genética , Animais , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Feminino , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Rim/fisiopatologia , Glomérulos Renais/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Estresse Oxidativo/fisiologia , Pâncreas/fisiopatologia , Peptidil Dipeptidase A/metabolismo , Sistema Renina-Angiotensina/fisiologia
15.
Life Sci ; 255: 117779, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32417374

RESUMO

OBJECTIVE: Kidney is the most common location of microangiopathy in diabetic patients, and we designed this study to investigate the effects of hirudin on renal microangiopathy in STZ-induced diabetes rats and in vitro. METHODS: We established a diabetes model by intraperitoneal injection of STZ and administered hirudin daily by subcutaneous injection. HE staining was used to assess kidney pathological changes. Western blot and immunochemistry was used to detect the protein expression. Glomerular endothelial cells (GEC) in normal rats were assessed by cell scratch test for migration ability and tubule formation experiment for angiogenesis ability. RESULTS: Compared with DN rats without any treatment, the serum creatinine, serum Cys C, 24-hour urine protein of DN rats with hirudin treatment were significantly decrease, the kidney/body weight and glomerular area of DN rats with hirudin treatment were all significantly decrease, and also significant improvement in renal pathology revealed by HE staining in DN rats after treating with hirudin. Moreover, we also found that hirudin coun not only significantly increase the prothrombin time and aivated partial thromboplastin time in DN rats, but also significantly decrease the expression of VEGF and TM-1 protein in kidney tissues of DN rats. In vitro, we found that high glucose could promote the migration and angiogensis of GEC, and significantly increased the expression of VEGF and Ang protein, but significantly decreased the expression of THBS1 and Arg1 protein. More importantly was that hirudin could inhibit the migration and angiogensis of GEC, and reversed HG-induced the expression of VEGF, Ang, THBS1 and Arg1 protein in GEC. In addition, we also found that hirudin could not only decrease HG-enhanced the activity of RhoA in GEC, but also decrease HG-enhanced the expression of p-MYPT1/MYPT1, p-p38/p38 protein in GEC. CONCLUSION: Hirudin reduces nephropathy microangiopathy in STZ-induced diabetes, and might be related to hirudin inhibiting glomerular endothelial cell migration and angiogenesis through Rho-kinase and subsequent p38MAPK/NF-kB signaling pathway.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Angiopatias Diabéticas/prevenção & controle , Nefropatias Diabéticas/prevenção & controle , Hirudinas/farmacologia , Neovascularização Patológica/prevenção & controle , Animais , Movimento Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/complicações , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Glucose/metabolismo , Glomérulos Renais/metabolismo , Masculino , NF-kappa B/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases Associadas a rho/metabolismo
16.
Am J Physiol Renal Physiol ; 318(5): F1237-F1245, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32223308

RESUMO

Changes in mitochondrial function are central to many forms of kidney disease, including acute injury, diabetic nephropathy, hypertension, and chronic kidney diseases. As such, there is an increasing need for reliable and fast methods for assessing mitochondrial respiratory function in renal cells. Despite being indispensable for many mechanistic studies, cultured cells or isolated mitochondria, however, often do not recapitulate in vivo or close to in vivo situations. Cultured and/or immortalized cells often change their bioenergetic profile and phenotype compared with in vivo or ex vivo situations, and isolated mitochondria are simply removed from their cellular milieu. This is especially important for extremely complex organs such as the kidney. Here, we report the development and validation of a new approach for the rapid assessment of mitochondrial oxygen consumption on freshly isolated glomeruli or proximal tubular fragments using Agilent SeaHorse XFe24 and XF96 Extracellular Flux Analyzers. We validated the technique in several healthy and diseased rodent models: the C57BL/6J mouse, the diabetic db/db mouse and matching db/+ control mouse, and the Dahl salt-sensitive rat. We compared the data to respiration from isolated mitochondria. The method can be adapted and used for the rapid assessment of mitochondrial oxygen consumption from any rodent model of the investigator's choice. The isolation methods presented here ensure viable and functional proximal tubular fragments and glomeruli, with a preserved cellular environment for studying mitochondrial function within the context of their surroundings and interactions.


Assuntos
Diabetes Mellitus/metabolismo , Metabolismo Energético , Hipertensão/metabolismo , Glomérulos Renais/metabolismo , Túbulos Renais Proximais/metabolismo , Mitocôndrias/metabolismo , Animais , Respiração Celular , Diabetes Mellitus/patologia , Modelos Animais de Doenças , Feminino , Hipertensão/patologia , Glomérulos Renais/patologia , Túbulos Renais Proximais/patologia , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/patologia , Consumo de Oxigênio , Ratos Endogâmicos Dahl
17.
Int J Mol Sci ; 21(7)2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32276523

RESUMO

It is widely known that glomerulonephritis (GN) often develops after the curing of an infection, a typical example of which is GN in children following streptococcal infections (poststreptococcal acute glomerulonephritis; PSAGN). On the other hand, the term "infection-related glomerulonephritis (IRGN)" has recently been proposed, because infections are usually ongoing at the time of GN onset in adult patients, particularly in older patients with comorbidities. However, there has been no specific diagnostic biomarker for IRGN, and diagnosis is based on the collection of several clinical and pathological findings and the exclusion of differential diagnoses. Nephritis-associated plasmin receptor (NAPlr) was originally isolated from the cytoplasmic fraction of group A streptococcus as a candidate nephritogenic protein for PSAGN and was found to be the same molecule as streptococcal glyceraldehyde-3-phosphate dehydrogenase and plasmin receptor. NAPlr deposition and related plasmin activity were observed with a similar distribution pattern in the glomeruli of patients with PSAGN. However, glomerular NAPlr deposition and plasmin activity could be observed not only in patients with PSAGN but also in patients with other glomerular diseases, in whom a preceding streptococcal infection was suggested. Furthermore, such glomerular staining patterns have been demonstrated in patients with IRGN induced by bacteria other than streptococci. This review discusses the recent advances in our understanding of the pathogenesis of bacterial IRGN, which is characterized by NAPlr and plasmin as key biomarkers.


Assuntos
Fibrinolisina/análise , Glomerulonefrite/diagnóstico , Receptores de Peptídeos/análise , Infecções Estreptocócicas/complicações , Infecções Bacterianas/complicações , Biomarcadores/análise , Glomerulonefrite/etiologia , Humanos , Glomérulos Renais/metabolismo
18.
PLoS One ; 15(4): e0231662, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32315336

RESUMO

Early detection of obesity-related glomerulopathy in humans is challenging as it might not be detected by routine biomarkers of kidney function. This study's aim was to use novel kidney biomarkers and contrast-enhanced ultrasound (CEUS) to evaluate the effect of obesity development and weight-loss on kidney function, perfusion, and injury in dogs. Sixteen healthy lean adult beagles were assigned randomly but age-matched to a control group (CG) (n = 8) fed to maintain a lean body weight (BW) for 83 weeks; or to a weight-change group (WCG) (n = 8) fed the same diet to induce obesity (week 0-47), to maintain stable obese weight (week 47-56) and to lose BW (week 56-83). At 8 time points, values of systolic blood pressure (sBP); serum creatinine (sCr); blood urea nitrogen (BUN); serum cystatin C (sCysC); urine protein-to-creatinine ratio (UPC); and urinary biomarkers of glomerular and tubular injury were measured. Glomerular filtration rate (GFR) and renal perfusion using CEUS were assayed (except for week 68). For CEUS, intensity- and time-related parameters representing blood volume and velocity were derived from imaging data, respectively. At 12-22% weight-gain, cortical time-to-peak, representing blood velocity, was shorter in the WCG vs. the CG. After 37% weight-gain, sCysC, UPC, glomerular and tubular biomarkers of injury, urinary immunoglobulin G and urinary neutrophil gelatinase-associated lipocalin, respectively, were higher in the WCG. sBP, sCr, BUN and GFR were not significantly different. After 23% weight-loss, all alterations were attenuated. Early weight-gain in dogs induced renal perfusion changes measured with CEUS, without hyperfiltration, preceding increased urinary protein excretion with potential glomerular and tubular injury. The combined use of routine biomarkers of kidney function, CEUS and site-specific urinary biomarkers might be valuable in assessing kidney health of individuals at risk for obesity-related glomerulopathy in a non-invasive manner.


Assuntos
Glomerulonefrite/metabolismo , Glomérulos Renais/metabolismo , Túbulos Renais/metabolismo , Obesidade/metabolismo , Ganho de Peso/genética , Animais , Biomarcadores/urina , Nitrogênio da Ureia Sanguínea , Meios de Contraste/farmacologia , Creatinina/sangue , Modelos Animais de Doenças , Cães , Taxa de Filtração Glomerular , Glomerulonefrite/etiologia , Glomerulonefrite/patologia , Glomerulonefrite/urina , Humanos , Glomérulos Renais/diagnóstico por imagem , Glomérulos Renais/lesões , Glomérulos Renais/patologia , Túbulos Renais/diagnóstico por imagem , Túbulos Renais/lesões , Túbulos Renais/patologia , Obesidade/complicações , Obesidade/diagnóstico por imagem , Obesidade/patologia , Ultrassonografia , Sistema Urinário/metabolismo , Sistema Urinário/patologia , Ganho de Peso/fisiologia , Perda de Peso/genética , Perda de Peso/fisiologia
19.
Proc Natl Acad Sci U S A ; 117(11): 6086-6091, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123080

RESUMO

Recombinant immunotoxins (RITs) are chimeric proteins composed of an Fv and a protein toxin being developed for cancer treatment. The Fv brings the toxin to the cancer cell, but most of the RITs do not reach the tumor and are removed by other organs. To identify cells responsible for RIT removal, and the pathway by which RITs reach these cells, we studied SS1P, a 63-kDa RIT that targets mesothelin-expressing tumors and has a short serum half-life. The major organs that remove RIT were identified by live mouse imaging of RIT labeled with FNIR-Z-759. Cells responsible for SS1P removal were identified by immunohistochemistry and intravital two-photon microscopy of kidneys of rats. The primary organ of SS1P removal is kidney followed by liver. In the kidney, SS1P passes through the glomerulus, is taken up by proximal tubular cells, and transferred to lysosomes. In the liver, macrophages are involved in removal. The short half-life of SS1P is due to its very rapid filtration by the kidney followed by degradation in proximal tubular cells of the kidney. In mice treated with SS1P, proximal tubular cells are damaged and albumin in the urine is increased. SS1P uptake by kidney is reduced by coadministration of l-lysine. Our data suggests that l-lysine administration to humans might prevent SS1P-mediated kidney damage, reduce albumin loss in urine, and alleviate capillary leak syndrome.


Assuntos
Albuminúria/patologia , Anticorpos Monoclonais/farmacocinética , Síndrome de Vazamento Capilar/patologia , Imunotoxinas/farmacocinética , Túbulos Renais Proximais/efeitos dos fármacos , Albuminúria/induzido quimicamente , Albuminúria/prevenção & controle , Albuminúria/urina , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/química , Anticorpos Monoclonais/toxicidade , Síndrome de Vazamento Capilar/induzido quimicamente , Síndrome de Vazamento Capilar/prevenção & controle , Síndrome de Vazamento Capilar/urina , Modelos Animais de Doenças , Feminino , Corantes Fluorescentes/química , Meia-Vida , Humanos , Imunotoxinas/administração & dosagem , Imunotoxinas/química , Imunotoxinas/toxicidade , Microscopia Intravital , Glomérulos Renais/metabolismo , Túbulos Renais Proximais/diagnóstico por imagem , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Lisina/administração & dosagem , Camundongos , Microscopia de Fluorescência , Neoplasias/tratamento farmacológico , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/toxicidade , Eliminação Renal/efeitos dos fármacos , Albumina Sérica/análise , Albumina Sérica/metabolismo , Coloração e Rotulagem
20.
J Am Soc Nephrol ; 31(4): 865-875, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32127409

RESUMO

BACKGROUND: In males with classic Fabry disease, the processes leading to the frequent outcome of ESKD are poorly understood. Defects in the gene encoding α-galactosidase A lead to accumulation of globotriaosylceramide (GL3) in various cell types. In the glomerular podocytes, accumulation of GL3 progresses with age. Of concern, podocytes are relatively resistant to enzyme replacement therapy and are poorly replicating, with little ability to compensate for cell loss. METHODS: In this study of 55 males (mean age 27 years) with classic Fabry disease genotype and/or phenotype, we performed unbiased quantitative morphometric electron microscopic studies of biopsied kidney samples from patients and seven living transplant donors (to serve as controls). We extracted clinical information from medical records and clinical trial databases. RESULTS: Podocyte GL3 volume fraction (proportion of podocyte cytoplasm occupied by GL3) increased with age up to about age 27, suggesting that increasing podocyte GL3 volume fraction beyond a threshold may compromise survival of these cells. GL3 accumulation was associated with podocyte injury and loss, as evidenced by increased foot process width (a generally accepted structural marker of podocyte stress and injury) and with decreased podocyte number density per glomerular volume. Worsening podocyte structural parameters (increasing podocyte GL3 volume fraction and foot process width) was also associated with increasing urinary protein excretion-a strong prognosticator of adverse renal outcomes in Fabry disease-as well as with decreasing GFR. CONCLUSIONS: Given the known association between podocyte loss and irreversible FSGS and global glomerulosclerosis, this study points to an important role for podocyte injury and loss in the progression of Fabry nephropathy and indicates a need for therapeutic intervention before critical podocyte loss occurs.


Assuntos
Doença de Fabry/metabolismo , Doença de Fabry/patologia , Podócitos/metabolismo , Podócitos/patologia , Triexosilceramidas/metabolismo , Adolescente , Adulto , Fatores Etários , Estudos de Casos e Controles , Criança , Pré-Escolar , Taxa de Filtração Glomerular , Humanos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Pessoa de Meia-Idade , Adulto Jovem
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