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1.
Toxicol Lett ; 321: 44-53, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31811911

RESUMO

This study was aimed to investigate the effect of prenatal ethanol exposure (PEE) on the susceptibility of offspring rats to glomerulosclerosis and to explore the mechanism. Pregnant Wistar rats were intragastrically administered ethanol (4g/kg·d) from gestational day (GD) 9 to GD 20, and the control group was given equal volume of normal saline. The offspring rats were all fed with high-fat diet after weaning, and were sacrificed at postnatal week 24 (PW24). The results revealed that the adult offspring kidneys in the male and female PEE groups exhibited higher glomerulosclerosis index and interstitial fibrosis index compared with the high-fat diet control groups, accompanied by elevated serum creatinine level. The protein expression of Nephrin and WT1, which were the marker genes of podocytes, was significantly decreased, whereas the protein expression of desmin and α-SMA, the marker genes of mesenchymal cells, was remarked enhanced in the male and female PEE groups. Compared with the high-fat diet control groups, the mRNA and protein expressions of renal angiotensin II receptor type 2 (AT2R) were decreased in the male PEE group, but increased in the female PEE group. PEE increased the mRNA and protein expressions of glucocorticoid (GC) activation system and inhibited the expression of insulin-like growth factor 1 (IGF1) signaling pathway in male offspring kidney; on the contrary, in female offspring kidney, PEE inhibited the mRNA and protein expression of glucocorticoid activation system and increased the expression of IGF1 signaling pathway. Taken together, PEE increased the susceptibility of the adult offspring to glomerulosclerosis, and the programming of renal AT2R or GC-IGF1 is respectively involved in the toxicity of PEE to the male or female offspring.


Assuntos
Dieta Hiperlipídica , Etanol/toxicidade , Glomerulonefrite/etiologia , Glomérulos Renais/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal , Transdução de Sinais/efeitos dos fármacos , Animais , Feminino , Fibrose , Regulação da Expressão Gênica , Idade Gestacional , Glomerulonefrite/genética , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Glucocorticoides/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Gravidez , Ratos Wistar , Receptor Tipo 2 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/metabolismo , Transdução de Sinais/genética
2.
PLoS One ; 14(12): e0220483, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31881024

RESUMO

E. coli associated Hemolytic Uremic Syndrome (epidemic hemolytic uremic syndrome, eHUS) caused by Shiga toxin-producing bacteria is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute kidney injury that cause acute renal failure in up to 65% of affected patients. We hypothesized that the mannose-binding lectin (MBL) pathway of complement activation plays an important role in human eHUS, as we previously demonstrated that injection of Shiga Toxin-2 (Stx-2) led to fibrin deposition in mouse glomeruli that was blocked by co-injection of the anti-MBL-2 antibody 3F8. However, the markers of platelet thrombosis in affected mouse glomeruli were not delineated. To investigate the effect of 3F8 on markers of platelet thrombosis, we used kidney sections from our mouse model (MBL-2+/+ Mbl-A/C-/-; MBL2 KI mouse). Mice in the control group received PBS, while mice in a second group received Stx-2, and those in a third group received 3F8 and Stx-2. Using double immunofluorescence (IF) followed by digital image analysis, kidney sections were stained for fibrin(ogen) and CD41 (marker for platelets), von-Willebrand factor (marker for endothelial cells and platelets), and podocin (marker for podocytes). Electron microscopy (EM) was performed on ultrathin sections from mice and human with HUS. Injection of Stx-2 resulted in an increase of both fibrin and platelets in glomeruli, while administration of 3F8 with Stx-2 reduced both platelet and fibrin to control levels. EM studies confirmed that CD41-positive objects observed by IF were platelets. The increases in platelet number and fibrin levels by injection of Stx-2 are consistent with the generation of platelet-fibrin thrombi that were prevented by 3F8.


Assuntos
Síndrome Hemolítico-Urêmica/metabolismo , Lectina de Ligação a Manose/metabolismo , Trombose/metabolismo , Lesão Renal Aguda/metabolismo , Animais , Plaquetas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Infecções por Escherichia coli/microbiologia , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Rim/metabolismo , Glomérulos Renais/metabolismo , Lectina de Ligação a Manose/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Toxina Shiga/metabolismo , Toxina Shiga II/metabolismo , Tromboembolia/metabolismo
3.
Phytomedicine ; 64: 153073, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31542661

RESUMO

BACKGROUND: Ginkgo biloba (Gb) extracts have been used as a traditional Chinese medicine. Gb contains flavonoids, which are considered to be its active ingredients and have been used in the treatment of a variety of diseases. However, few scientific research studies on the side effects of flavonoid in Gb have been reported. PURPOSE: The present study aimed to investigate the effect of bilobetin on the kidney of Sprague-Dawley (SD) rats. STUDY DESIGN AND RESULT: In this study, rats were injected with 50 mg/kg of bilobetin, a biflavone isolated from Gb, for 7 days and aristolochic acid was used as positive controls. The results showed that the body weight and urine output of the rats were dramatically decreased, and urinary protein increased after the intraperitoneal injection of bilobetin compared with the control group. Bilobetin treatment showed vacuolar degeneration in the renal tubular epithelium, glomerular atrophy by histostaining, and podocyte fusion by electron microscopy. This study further showed that bilobetin promoted the trafficking of aquaporin 2 (AQP-2) onto the plasma membrane to achieve the function of urine concentration by in vivo study in rats and in vitro study in IMCD-3 cells. The redistribution of AQP-2 is due to increased expression of cGMP in IMCD-3 cells, which in turn promoted the phosphorylation of AQP-2 at site Ser-256. The proteinuria caused by bilobetin may be attributed to podocyte cell cycle arrest at G2/M transition, which is may associated with AKT and MAPK signaling. CONCLUSIONS: The current study showed that bilobetin has some side effects on kidneys at a dose of 50 mg/kg in SD rats and provides insight into the potential detrimental effects of monomeric ingredients in Gb.


Assuntos
Lesão Renal Aguda/induzido quimicamente , Lesão Renal Aguda/patologia , Aquaporina 2/metabolismo , Flavonoides/efeitos adversos , Podócitos/efeitos dos fármacos , Lesão Renal Aguda/metabolismo , Animais , Aquaporina 2/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , GMP Cíclico/metabolismo , Medicamentos de Ervas Chinesas/efeitos adversos , Medicamentos de Ervas Chinesas/química , Flavonoides/administração & dosagem , Células HEK293 , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Glomérulos Renais/metabolismo , Masculino , Extratos Vegetais/efeitos adversos , Extratos Vegetais/química , Podócitos/metabolismo , Podócitos/patologia , Proteinúria/induzido quimicamente , Ratos Sprague-Dawley
4.
Int J Mol Sci ; 20(17)2019 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-31480394

RESUMO

Feeding rats with high-fat diet (HFD) with a single streptozotocin (STZ) injection induced obesity, slightly elevated fasting blood glucose and impaired glucose and insulin tolerance, and caused cardiac hypertrophy and mild diastolic dysfunction as published before by Koncsos et al. in 2016. Here we aimed to explore the renal consequences in the same groups of rats. Male Long-Evans rats were fed normal chow (CON; n = 9) or HFD containing 40% lard and were administered STZ at 20 mg/kg (i.p.) at week four (prediabetic rats, PRED, n = 9). At week 21 blood and urine samples were taken and kidney and liver samples were collected for histology, immunohistochemistry and for analysis of gene expression. HFD and STZ increased body weight and visceral adiposity and plasma leptin concentration. Despite hyperleptinemia, plasma C-reactive protein concentration decreased in PRED rats. Immunohistochemistry revealed elevated collagen IV protein expression in the glomeruli, and Lcn2 mRNA expression increased, while Il-1ß mRNA expression decreased in both the renal cortex and medulla in PRED vs. CON rats. Kidney histology, urinary protein excretion, plasma creatinine, glomerular Feret diameter, desmin protein expression, and cortical and medullary mRNA expression of TGF-ß1, Nrf2, and PPARγ were similar in CON and PRED rats. Reduced AMPKα phosphorylation of the autophagy regulator Akt was the first sign of liver damage, while plasma lipid and liver enzyme concentrations were similar. In conclusion, glomerular collagen deposition and increased lipocalin-2 expression were the early signs of kidney injury, while most biomarkers of inflammation, oxidative stress and fibrosis were negative in the kidneys of obese, prediabetic rats with mild heart and liver injury.


Assuntos
Colágeno/metabolismo , Glomérulos Renais/lesões , Glomérulos Renais/metabolismo , Lipocalina-2/metabolismo , Obesidade/metabolismo , Estado Pré-Diabético/metabolismo , Tecido Adiposo/metabolismo , Animais , Biomarcadores/metabolismo , Peso Corporal , Dieta Hiperlipídica , Fibrose , Regulação da Expressão Gênica , Inflamação/genética , Inflamação/patologia , Glomérulos Renais/patologia , Lipídeos/sangue , Fígado/enzimologia , Fígado/patologia , Fígado/fisiopatologia , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Obesidade/sangue , Estresse Oxidativo/genética , Fosforilação , Fosfosserina/metabolismo , Estado Pré-Diabético/sangue , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Long-Evans , Estreptozocina
6.
Molecules ; 24(15)2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31390847

RESUMO

Diabetic kidney disease develops in approximately 40% of diabetic patients and is a major cause of chronic kidney diseases (CKD) and end stage kidney disease (ESKD) worldwide. Hydrogen sulfide (H2S), the third gasotransmitter after nitric oxide (NO) and carbon monoxide (CO), is synthesized in nearly all organs, including the kidney. Though studies on H2S regulation of renal physiology and pathophysiology are still in its infancy, emerging evidence shows that H2S production by renal cells is reduced under disease states and H2S donors ameliorate kidney injury. Specifically, aberrant H2S level is implicated in various renal pathological conditions including diabetic nephropathy. This review presents the roles of H2S in diabetic renal disease and the underlying mechanisms for the protective effects of H2S against diabetic renal damage. H2S may serve as fundamental strategies to treat diabetic kidney disease. These H2S treatment modalities include precursors for H2S synthesis, H2S donors, and natural plant-derived compounds. Despite accumulating evidence from experimental studies suggests the potential role of the H2S signaling pathway in the treatment of diabetic nephropathy, these results need further clinical translation. Expanding understanding of H2S in the kidney may be vital to translate H2S to be a novel therapy for diabetic renal disease.


Assuntos
Sulfeto de Hidrogênio/metabolismo , Animais , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Avaliação Pré-Clínica de Medicamentos , Fibrose , Humanos , Sulfeto de Hidrogênio/química , Sulfeto de Hidrogênio/farmacologia , Sulfeto de Hidrogênio/uso terapêutico , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Redes e Vias Metabólicas/efeitos dos fármacos , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Oxigênio/metabolismo , Podócitos/metabolismo , Podócitos/patologia , Sistema Renina-Angiotensina
7.
Life Sci ; 234: 116738, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31398418

RESUMO

AIMS: Oxidative stress has been linked to the development and progression of diabetic nephropathy (DN). The present study evaluated whether the dipeptidyl peptidase-4 inhibitor sitagliptin attenuates glomerular lesions and oxidative stress evoked by chronic hyperglycemia, by a mechanism independent of insulin secretion and glycemia normalization. MAIN METHODS: A rat model of DN caused by streptozotocin injection was established and the effects of sitagliptin (5 mg/kg/day) were evaluated after two weeks of treatment. KEY FINDINGS: Sitagliptin treatment did not change body weight, glycemic and lipid profiles. However, histopathological observation revealed that sitagliptin attenuates diabetes-induced glomerular lesions on diabetic rats. Sitagliptin also ameliorated the increase in DPP-4 content and promoted the stabilization of GLP-1 in the diabetic kidney. Furthermore, sitagliptin treatment significantly attenuated the increase of free-radical formation and the decrease of antioxidant defenses, attenuating therefore the oxidative stress in the kidneys of diabetic animals. SIGNIFICANCE: The results suggest that sitagliptin treatment alleviates kidney oxidative stress in type 1 diabetic rats, which could play a key role in reducing the progression of DN.


Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Glomérulos Renais/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fosfato de Sitagliptina/uso terapêutico , Animais , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Inibidores da Dipeptidil Peptidase IV/farmacologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Ratos , Ratos Wistar , Fosfato de Sitagliptina/farmacologia
8.
Nat Commun ; 10(1): 3656, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409793

RESUMO

In this work we model the glomerular filtration barrier, the structure responsible for filtering the blood and preventing the loss of proteins, using human podocytes and glomerular endothelial cells seeded into microfluidic chips. In long-term cultures, cells maintain their morphology, form capillary-like structures and express slit diaphragm proteins. This system recapitulates functions and structure of the glomerulus, including permselectivity. When exposed to sera from patients with anti-podocyte autoantibodies, the chips show albuminuria proportional to patients' proteinuria, phenomenon not observed with sera from healthy controls or individuals with primary podocyte defects. We also show its applicability for renal disease modeling and drug testing. A total of 2000 independent chips were analyzed, supporting high reproducibility and validation of the system for high-throughput screening of therapeutic compounds. The study of the patho-physiology of the glomerulus and identification of therapeutic targets are also feasible using this chip.


Assuntos
Glomérulos Renais/metabolismo , Dispositivos Lab-On-A-Chip , Nefrite Hereditária/metabolismo , Albuminas/metabolismo , Albuminúria/tratamento farmacológico , Albuminúria/metabolismo , Células Imobilizadas/química , Células Imobilizadas/metabolismo , Células Endoteliais/química , Células Endoteliais/metabolismo , Humanos , Glomérulos Renais/química , Glomérulos Renais/efeitos dos fármacos , Masculino , Nefrite Hereditária/tratamento farmacológico , Podócitos/química , Podócitos/metabolismo
9.
Hypertension ; 74(4): 864-871, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31422689

RESUMO

It is well known that high protein intake increases glomerular filtration rate. Evidence from several studies indicated that NO and tubuloglomerular feedback (TGF) mediate the effect. However, a recent study with a neuronal NO synthase-α knockout model refuted this mechanism and concluded that neither neuronal NO synthase nor TGF response is involved in the protein-induced hyperfiltration. To examine the discrepancy, this study tested a hypothesis that neuronal NO synthase-ß in the macula densa mediates the high-protein diet-induced glomerular hyperfiltration via TGF mechanism. We examined the effects of high protein intake on NO generation at the macula densa, TGF response, and glomerular filtration rate in wild-type and macula densa-specific neuronal NO synthase KO mice. In wild-type mice, high-protein diet increased kidney weight, glomerular filtration rate, and renal blood flow, while reduced renal vascular resistance. TGF response in vivo and in vitro was blunted, and NO generation in the macula densa was increased following high-protein diet, associated with upregulations of neuronal NO synthase-ß expression and phosphorylation at Ser1417. In contrast, these high-protein diet-induced changes in NO generation at the macula densa, TGF response, renal blood flow, and glomerular filtration rate in wild-type mice were largely attenuated in macula densa-specific neuronal NO synthase KO mice. In conclusion, we demonstrated that high-protein diet-induced glomerular hyperfiltration is dependent on neuronal NO synthase ß in the macula densa via TGF response.


Assuntos
Retroalimentação Fisiológica/fisiologia , Taxa de Filtração Glomerular/fisiologia , Nefropatias/metabolismo , Glomérulos Renais/metabolismo , Rim/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Animais , Dieta Rica em Proteínas , Masculino , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo I/genética
10.
Int J Mol Sci ; 20(15)2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31362427

RESUMO

Amplification of oxidative stress is present since the early stages of chronic kidney disease (CKD), holding a key position in the pathogenesis of renal failure. Induction of renal pro-oxidant enzymes with excess generation of reactive oxygen species (ROS) and accumulation of dityrosine-containing protein products produced during oxidative stress (advanced oxidation protein products-AOPPs) have been directly linked to podocyte damage, proteinuria, and the development of focal segmental glomerulosclerosis (FSGS) as well as tubulointerstitial fibrosis. Vascular oxidative stress is considered to play a critical role in CKD progression, and ROS are potential mediators of the impaired myogenic responses of afferent renal arterioles in CKD and impaired renal autoregulation. Both oxidative stress and inflammation are CKD hallmarks. Oxidative stress promotes inflammation via formation of proinflammatory oxidized lipids or AOPPs, whereas activation of nuclear factor κB transcription factor in the pro-oxidant milieu promotes the expression of proinflammatory cytokines and recruitment of proinflammatory cells. Accumulating evidence implicates oxidative stress in various clinical models of CKD, including diabetic nephropathy, IgA nephropathy, polycystic kidney disease as well as the cardiorenal syndrome. The scope of this review is to tackle the issue of oxidative stress in CKD in a holistic manner so as to provide a future framework for potential interventions.


Assuntos
Estresse Oxidativo , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/metabolismo , Albuminúria/etiologia , Albuminúria/metabolismo , Animais , Biomarcadores , Gerenciamento Clínico , Modelos Animais de Doenças , Progressão da Doença , Fibrose , Humanos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Microvasos/metabolismo , Microvasos/patologia , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/terapia
11.
PLoS One ; 14(7): e0219403, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31291349

RESUMO

IgA nephropathy, a common chronic kidney disease, has various possible outcomes. Therefore, the identification of novel prognostic biomarkers is needed. To this purpose, we used gas chromatography mass spectrometry to search for metabolites capable of predicting the phenotypes of IgA nephropathy in hyper IgA (HIGA) mice, an established model mice for IgA nephropathy. We measured the plasma metabolite levels in 12- and 22-week-old mice, prior to the manifestation of IgA nephropathy phenotypes, and statistically investigated the associations between these metabolites and the phenotypes of IgA nephropathy, such as the urine protein levels and histological phenotypes of the kidney at 32 weeks. We observed that in plasma samples collected from 12- and 22-week-old HIGA mice, the urinary protein levels were significantly associated with 8 and 10 metabolites, the glomerular cellular component levels were significantly associated with 8 and 7 metabolites, and the mesangial substrate levels were significantly associated with 8 and 8 metabolites, respectively. Among the candidate metabolites associated with the phenotypes of IgA nephropathy, coniferyl alcohol levels were significantly higher in HIGA mice at all of the 12, 22, and 32 weeks of age. Since this study was an observational study, we could not elucidate the underlying mechanisms; however, we were able to identify new candidate metabolites, such as coniferyl alcohol, as being potentially involved in the pathogenesis of IgA nephropathy. These results might help to develop novel laboratory tests and therapeutic reagents for IgA nephropathy in the future.


Assuntos
Glomerulonefrite por IGA/sangue , Imunoglobulina A/sangue , Metaboloma , Animais , Modelos Animais de Doenças , Cromatografia Gasosa-Espectrometria de Massas , Mesângio Glomerular/metabolismo , Mesângio Glomerular/patologia , Glomerulonefrite por IGA/patologia , Humanos , Rim/metabolismo , Rim/patologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Camundongos , Fenótipo
13.
Biomed Pharmacother ; 118: 109175, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31351423

RESUMO

Increasing research indicates that hyperglycemia plays a crucial role in the progression of diabetic nephropathy (DN); however, effective treatment for preventing or slowing DN progression are seriously lacking. Although salidroside (SAL) has been demonstrated to have a positive anti-diabetic effect, the cellular mechanisms remain unclear. FG-4592, a novel prolyl hydroxylase inhibitor, was used to regulate HIF-1α and HIF-2α expression. The present study aimed to explore the underlying mechanisms of SAL and FG-4592 on high glucose (HG)-induced rat glomerular endothelial cells (rGECs) injury. HG-cultured rGECs were used to induce a diabetic environment. An MTT assay, RT-qPCR, Western blot, flow cytometry, and immunofluorescent staining were performed to investigate the effects of SAL on HG-induced rGECs injury. FG-4592 and SAL protected rGECs against HG-induced injury by increasing cellular viability and reducing the cell apoptosis rate. SAL and FG-4592 downregulated PHD-2 expression and upregulated HIF-1α and HIF-2α expression. In conclusion, our findings suggest that SAL and FG-4592 ameliorate HG-induced rGEC injury by upregulating HIF expression, indicating that SAL and FG-4592 might be favorable for further DN-treatment.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Células Endoteliais/efeitos dos fármacos , Glucose/toxicidade , Glucosídeos/farmacologia , Glicina/análogos & derivados , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Isoquinolinas/farmacologia , Glomérulos Renais/efeitos dos fármacos , Fenóis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Glucosídeos/administração & dosagem , Glicina/administração & dosagem , Glicina/farmacologia , Isoquinolinas/administração & dosagem , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Fenóis/administração & dosagem , Estabilidade Proteica , Ratos , Regulação para Cima
14.
Dev Cell ; 50(1): 102-116.e6, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31265809

RESUMO

The renal corpuscle of the kidney comprises a glomerular vasculature embraced by podocytes and supported by mesangial myofibroblasts, which ensure plasma filtration at the podocyte-generated slit diaphragm. With a spectrum of podocyte-expressed gene mutations causing chronic disease, an enhanced understanding of podocyte development and function to create relevant in vitro podocyte models is a clinical imperative. To characterize podocyte development, scRNA-seq was performed on human fetal kidneys, identifying distinct transcriptional signatures accompanying the differentiation of functional podocytes from progenitors. Interestingly, organoid-generated podocytes exhibited highly similar, progressive transcriptional profiles despite an absence of the vasculature, although abnormal gene expression was pinpointed in late podocytes. On transplantation into mice, organoid-derived podocytes recruited the host vasculature and partially corrected transcriptional profiles. Thus, human podocyte development is mostly intrinsically regulated and vascular interactions refine maturation. These studies support the application of organoid-derived podocytes to model disease and to restore or replace normal kidney functions.


Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes Induzidas/citologia , Glomérulos Renais/citologia , Organoides/citologia , Podócitos/citologia , Análise de Célula Única/métodos , Células Cultivadas , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Glomérulos Renais/metabolismo , Organoides/metabolismo , Podócitos/metabolismo
15.
PLoS One ; 14(6): e0217585, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31220088

RESUMO

BACKGROUND: Renal fibrosis is the result of the interaction of cellular and molecular pathways, which is induced by sustained glomerular injury and involves the podocytes and multiple profibrotic factors. In this study, we investigated the correlation of the mRNA expression of podocyte proteins and profibrotic factors with renal fibrosis measured in renal biopsies of patients with primary and secondary glomerulopathies. METHODS: Eighty-four adult patients with primary or secondary glomerular diseases and 12 controls were included. Demographic and clinical data were collected. Seventy-two percent of the renal biopsies were done less than one year from clinical disease manifestation. The quantification of the podocyte-associated mRNAs of alpha-actinin-4, podocin, and podocalyxin, as well as of the profibrotic factors TGF-ß1, CTGF, and VEGF-A were quantified by real-time polymerase chain reaction. The percent positive area of renal fibrosis was measured by immunohistochemistry staining, using anti-CTGF and anti-HHF35 antibodies and unpolarized Sirius Red. Correlations between the expression of tissue mRNAs and the positive area of fibrosis for the measured markers were made by Spearman's rank correlation coefficient. RESULTS: In relation to control biopsies, podocyte-specific proteins were downregulated in podocytopathies, in proliferative nephritis, in diabetic kidney disease (DRD), and in IgA nephropathy (IgAN). Messenger RNA of TGF-ß1, CTGF, and VEGF-A was upregulated in patients with podocytopathies and in DRD but not in proliferative nephritis and IgAN. Tissue mRNA expression of TGF-ß1, CTGF, and VEGF-A were strongly correlated with renal fibrosis, as measured by HHF35; however, the correlation, albeit significant, was moderate for Sirius Red and weak for CTGF. The percent positive area of renal fibrosis measured by Sirius Red was similar between podocytopathies and DRD and significantly higher in podocytopathies compared to IgAN or proliferative nephritis. CONCLUSIONS: In patients with glomerular diseases, the mRNA of TGF-ß1, CTGF, and VEGF-A correlated positively with the extent of renal fibrosis, and the positive area of fibrosis was larger in the podocytopathies and in DRD as measured by Sirius Red. The pathways connecting podocyte damage and activation of profibrotic factors to kidney tissue fibrosis need to be better investigated.


Assuntos
Glomérulos Renais/patologia , Podócitos/patologia , Adulto , Biomarcadores/metabolismo , Biópsia , Fator de Crescimento do Tecido Conjuntivo/genética , Feminino , Humanos , Glomérulos Renais/metabolismo , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Fator de Crescimento Transformador beta1/genética , Fator A de Crescimento do Endotélio Vascular/genética
16.
J Diabetes Res ; 2019: 1602892, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31179338

RESUMO

This study was aimed at investigating the synergistical protective effects of Astragalus membranaceus (AG) and Panax notoginseng (NG) on podocyte injury in diabetic rats. Diabetes was induced in rats by a single intraperitoneal injection of streptozotocin at 55 mg/kg. Diabetic rats were then orally administrated with losartan, AG, NG, and AG plus NG (2 : 1) for 12 weeks. Albuminuria, biochemical markers, renal histopathology, and podocyte number per glomerulus were measured. Podocyte apoptosis was determined by triple immunofluorescence labeling including TUNEL assay, WT1, and DAPI. Renal expression of nephrin, α-dystroglycan, Bax, Bcl-xl, and Nox4 was evaluated by immunohistochemistry, western blot, and RT-PCR. AG plus NG ameliorated albuminuria, renal histopathology, and podocyte foot process effacement to a greater degree than did AG or NG alone. The number of podocytes per glomerulus, as well as renal expression of nephrin, α-dystroglycan, and Bcl-xl, was decreased, while podocyte apoptosis, as well as renal expression of Bax and Nox4, was increased in diabetic rats. All of these abnormalities were partially restored by AG plus NG to a greater degree than did AG or NG alone. In conclusion, AG and NG synergistically ameliorated diabetic podocyte injury partly through upregulation of nephrin, α-dystroglycan, and Bcl-xl, as well as downregulation of Bax and Nox4. These findings might provide a novel treatment combination for DN.


Assuntos
Astragalus propinquus/química , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Glomérulos Renais/efeitos dos fármacos , Panax notoginseng/química , Extratos Vegetais/farmacologia , Podócitos/efeitos dos fármacos , Albuminúria/metabolismo , Animais , Apoptose , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/metabolismo , Glomérulos Renais/metabolismo , Masculino , Podócitos/metabolismo , Substâncias Protetoras/farmacologia , Ratos , Ratos Sprague-Dawley , Estreptozocina
17.
PLoS One ; 14(6): e0217569, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31188898

RESUMO

There are controversies whether Minimal Change Disease (MCD) and Focal and Segmental Glomerulosclerosis (FSGS) are distinct glomerular lesions or different manifestations within the same spectrum of diseases. The uPAR (urokinase-type plasminogen activator receptor) and some slit diaphragm proteins may be altered in FSGS glomeruli and may function as biomarkers of the disease in renal biopsies. Thus, this study aims to evaluate the diagnostic potential of uPAR and glomerular proteins for differentiation between MCD and FSGS in renal pediatric biopsy. Renal biopsies from 50 children between 2 and 18 years old were selected, with diagnosis of MCD (n = 29) and FSGS (n = 21). Control group consisted of pediatric autopsies (n = 15) from patients younger than 18 years old, with no evidences of renal dysfunction. In situ expressions of WT1, nephrin, podocin and uPAR were evaluated by immunoperoxidase technique. Renal biopsy of patients with MCD and FSGS expressed fewer WT1 (p≤0.0001, F = 19.35) and nephrin (p<0.0001; H = 21.54) than patients in the control group. FSGS patients expressed fewer podocin than control (p<0.0359, H = 6.655). FSGS cases expressed more uPAR than each of control and MCD (p = 0.0019; H = 12.57) and there was a positive and significant correlation between nephrin and podocin (p = 0.0026, rS = 0.6502) in these cases. Podocin had sensitivity of 73.3% and specificity of 86.7% (p = 0.0068) and uPAR had sensitivity of 78.9% and specificity of 73.3% (p = 0.0040) for diagnosis of FSGS patients. The main limitation of the study is the limited number of cases due to the difficulty in performing biopsy in pediatric patients. Podocin and uPAR are good markers for FSGS and differentiate these cases from MCD, reinforcing the theory of distinct glomerular diseases. These findings suggest that podocin and uPAR can be used as biomarkers in the routine analysis of renal biopsies in cases of podocytopathies when the lesion (sclerosis) is not sampled.


Assuntos
Glomerulosclerose Segmentar e Focal/diagnóstico , Peptídeos e Proteínas de Sinalização Intracelular/genética , Glomérulos Renais/metabolismo , Proteínas de Membrana/genética , Nefrose Lipoide/diagnóstico , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Adolescente , Autopsia , Biomarcadores/metabolismo , Biópsia , Estudos de Casos e Controles , Criança , Pré-Escolar , Diagnóstico Diferencial , Feminino , Expressão Gênica , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Glomérulos Renais/patologia , Masculino , Proteínas de Membrana/metabolismo , Nefrose Lipoide/genética , Nefrose Lipoide/metabolismo , Nefrose Lipoide/patologia , Valor Preditivo dos Testes , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Proteínas WT1/genética , Proteínas WT1/metabolismo
18.
J Biol Chem ; 294(28): 10773-10788, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31152064

RESUMO

Nephrin is an immunoglobulin-type cell-adhesion molecule with a key role in the glomerular interpodocyte slit diaphragm. Mutations in the nephrin gene are associated with defects in the slit diaphragm, leading to early-onset nephrotic syndrome, typically resistant to treatment. Although the endocytic trafficking of nephrin is essential for the assembly of the slit diaphragm, nephrin's specific endocytic motifs remain unknown. To search for endocytic motifs, here we performed a multisequence alignment of nephrin and identified a canonical YXXØ-type motif, Y1139RSL, in the nephrin cytoplasmic tail, expressed only in primates. Using site-directed mutagenesis, various biochemical methods, single-plane illumination microscopy, a human podocyte line, and a human nephrin-expressing zebrafish model, we found that Y1139RSL is a novel endocytic motif and a structural element for clathrin-mediated nephrin endocytosis that functions as a phosphorylation-sensitive signal. We observed that Y1139RSL motif-mediated endocytosis helps to localize nephrin to specialized plasma membrane domains in podocytes and is essential for normal foot process organization into a functional slit diaphragm between neighboring foot processes in zebrafish. The importance of nephrin Y1139RSL for healthy podocyte development was supported by population-level analyses of genetic variations at this motif, revealing that such variations are very rare, suggesting that mutations in this motif have autosomal-recessive negative effects on kidney health. These findings expand our understanding of the mechanism underlying nephrin endocytosis and may lead to improved diagnostic tools or therapeutic strategies for managing early-onset, treatment-resistant nephrotic syndrome.


Assuntos
Glomérulos Renais/metabolismo , Proteínas de Membrana/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Movimento Celular , Clatrina/metabolismo , Embrião não Mamífero/metabolismo , Endocitose , Humanos , Glomérulos Renais/ultraestrutura , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Morfolinos/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação , Podócitos/citologia , Podócitos/metabolismo , Peixe-Zebra/crescimento & desenvolvimento
19.
J Pharmacol Exp Ther ; 370(2): 206-217, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31118214

RESUMO

Dual-specificity protein phosphatase 5 (DUSP5) is a member of the tyrosine-threonine phosphatase family with the ability to dephosphorylate and inactivate extracellular signal-related kinase (ERK). The present study investigates whether knockout (KO) of Dusp5 improves renal hemodynamics and protects against hypertension-induced renal injury. The renal expression of DUSP5 was reduced, and the levels of phosphorylated (p) ERK1/2 and p-protein kinase C (PKC) α were elevated in the KO rats. KO of Dusp5 enhanced the myogenic tone of the renal afferent arteriole and interlobular artery in vitro with or without induction of deoxycorticosterone acetate-salt hypertension. Inhibition of ERK1/2 and PKC diminished the myogenic response to a greater extent in Dusp5 KO rats. Autoregulation of renal blood flow was significantly impaired in hypertensive wild-type (WT) rats but remained intact in Dusp5 KO animals. Proteinuria was markedly decreased in hypertensive KO versus WT rats. The degree of glomerular injury was reduced, and the expression of nephrin in the glomerulus was higher in hypertensive Dusp5 KO rats. Renal fibrosis and medullary protein cast formation were attenuated in hypertensive Dusp5 KO rats in association with decreased expression of monocyte chemoattractant protein 1, transforming growth factor-ß1, matrix metalloproteinase (MMP) 2, and MMP9. These results indicate that KO of Dusp5 protects against hypertension-induced renal injury, at least in part, by maintaining the myogenic tone of the renal vasculature and extending the range of renal blood flow autoregulation to higher pressures, which diminish glomerular injury, protein cast formation, macrophage infiltration, and epithelial-mesenchymal transformation in the kidney. SIGNIFICANCE STATEMENT: Dual-specificity protein phosphatase 5 (DUSP5) is a tyrosine-threonine phosphatase that inactivates extracellular signal-related kinase (ERK). We previously reported that knockout (KO) of Dusp5 enhanced the myogenic response and autoregulation in the cerebral circulation. The present study investigates whether KO of DUSP5 improves renal hemodynamics and protects against hypertension-induced renal injury. Downregulation of DUSP5 enhanced the myogenic tone of renal arteriole and artery and autoregulation of renal blood flow in association with reduced proteinuria, glomerular injury, and interstitial fibrosis after the induction of hypertension. Inhibition of ERK1/2 and protein kinase C diminished the myogenic response to a greater extent in Dusp5 KO rats. These results suggest that DUSP5 might be a viable drug target for the treatment of hypertension nephropathy.


Assuntos
Fosfatases de Especificidade Dupla/deficiência , Fosfatases de Especificidade Dupla/genética , Técnicas de Inativação de Genes , Hipertensão Renal/genética , Nefrite/genética , Animais , Quimiocina CCL2/metabolismo , Regulação para Baixo/genética , Transição Epitelial-Mesenquimal/genética , Fibrose , Regulação Enzimológica da Expressão Gênica/genética , Hemodinâmica/genética , Hipertensão Renal/metabolismo , Hipertensão Renal/patologia , Hipertensão Renal/fisiopatologia , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Metaloproteinases da Matriz/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Desenvolvimento Muscular/genética , Nefrite/metabolismo , Nefrite/patologia , Nefrite/fisiopatologia , Proteína Quinase C/metabolismo , Ratos , Fluxo Sanguíneo Regional/genética , Fator de Crescimento Transformador beta/metabolismo
20.
Toxicol Lett ; 311: 17-26, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31039417

RESUMO

Prenatal ethanol exposure (PEE) causes intrauterine growth retardation (IUGR), and the occurrence of glomerulosclerosis is closely related to IUGR. This study aimed to confirm the kidney toxic effect of PEE and explore its intrauterine programming mechanism in female offspring. The Wistar female fetuses on gestational day (GD) 20 and the adult offspring at postnatal week 24 were anesthetized and decapitated. The adult offspring kidneys in the PEE group displayed glomerular hyperplasia and glomerulosclerosis. Blood urea nitrogen (BUN) and the BUN / Serum creatinine (Scr) concentration ratio in the PEE group was increased significantly compared to the control group (P<0.01, P<0.05). Meanwhile, the renal glucocorticoid-activation system was inhibited, whereas the insulin-like growth factor 1 (IGF1) signaling pathway was activated in the female adult offspring of the PEE group. In the fetal kidney of the PEE group, pathological observation showed kidney dysplasia, and the gene expression of the glial-cell-line-derived neurotrophic factor/tyrosine kinase receptor (GDNF/c-Ret) signaling pathway was reduced compared to that of the control group. Moreover, the glucocorticoid-activation system was activated, whereas the IGF1 signaling pathway was inhibited in the fetal kidneys of the PEE group. In conclusion, PEE caused fetal kidney dysplasia and adult glomerulosclerosis in the female offspring rats, and the intrauterine programming alteration of glucocorticoid-insulin-like growth factor 1 (GC-IGF1) axis might be involved in fetal-originated glomerulosclerosis.


Assuntos
Etanol/toxicidade , Glucocorticoides/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Nefropatias/induzido quimicamente , Glomérulos Renais/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal , Animais , Feminino , Desenvolvimento Fetal/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Idade Gestacional , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator de Crescimento Insulin-Like I/genética , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Gravidez , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Ratos Wistar , Fatores Sexuais , Transdução de Sinais/efeitos dos fármacos
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