RESUMO
Membranous nephropathy (MN) is a common immune-mediated glomerular disease that requires the development of safe and highly effective therapies. Celastrol (CLT) has shown promise as a therapeutic molecule candidate, but its clinical use is currently limited due to off-target toxicity. Given that excess levels of reactive oxygen species (ROS) contributing to podocyte damage is a key driver of MN progression to end-stage renal disease, we rationally designed ROS-responsive cationic polymeric nanoparticles (PPS-CPNs) with a well-defined particle size and surface charge by employing poly(propylene sulfide)-polyethylene glycol (PPS-PEG) and poly(propylene sulfide)-polyethylenimine (PPS-PEI) to selectively deliver CLT to the damaged glomerulus for MN therapy. Experimental results show that PPS-CPNs successfully crossed the fenestrated endothelium, accumulated in the glomerular basement membrane (GBM), and were internalized by podocytes where rapid drug release was triggered by the overproduction of ROS, thereby outperforming nonresponsive CLT nanotherapy to alleviate subepithelial immune deposits, podocyte foot process effacement, and GBM expansion in a rat MN model. Moreover, the ROS-responsive CLT nanotherapy was associated with significantly lower toxicity to major organs than free CLT. These results suggest that encapsulating CLT into PPS-CPNs can improve efficacy and reduce toxicity as a promising treatment option for MN.
Assuntos
Glomerulonefrite Membranosa , Nanopartículas , Triterpenos Pentacíclicos , Podócitos , Espécies Reativas de Oxigênio , Animais , Espécies Reativas de Oxigênio/metabolismo , Nanopartículas/química , Glomerulonefrite Membranosa/tratamento farmacológico , Glomerulonefrite Membranosa/patologia , Ratos , Triterpenos Pentacíclicos/química , Triterpenos Pentacíclicos/farmacologia , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Polietilenoglicóis/química , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/patologia , Glomérulos Renais/metabolismo , Ratos Sprague-Dawley , Humanos , Masculino , Polímeros/química , Polímeros/farmacologia , Sulfetos/química , Sulfetos/farmacologia , Sulfetos/uso terapêutico , Polietilenoimina/química , Portadores de Fármacos/químicaRESUMO
INTRODUCTION: Immune checkpoint inhibitors (ICIs) induce acute interstitial nephritis (AIN) in 2-5% of patients, with a clearly higher incidence when they are combined with platinum derivatives. Unfortunately, suitable disease models and non-invasive biomarkers are lacking. To fill this gap in our understanding, we investigated the renal effects of cisplatin and anti-PD-L1 antibodies in mice, assessing PD-1 renal expression and cytokine levels in mice with AIN, and then we compared these findings with those in AIN-diagnosed cancer patients. METHODS: Twenty C57BL6J mice received 200 µg of anti-PD-L1 antibody and 5 mg/kg cisplatin intraperitoneally and were compared with those receiving cisplatin (n = 6), anti-PD-L1 (n = 7), or saline (n = 6). After 7 days, the mice were euthanized. Serum and urinary concentrations of TNFα, CXCL10, IL-6, and MCP-1 were measured by Luminex. The kidney sections were stained to determine PD-1 tissue expression. Thirty-nine cancer patients with AKI were enrolled (AIN n = 33, acute tubular necrosis (ATN) n = 6), urine MCP-1 (uMCP-1) was measured, and kidney sections were stained to assess PD-1 expression. RESULTS: Cisplatin and anti PD-L1 treatment led to 40% AIN development (p = 0.03) in mice, accompanied by elevated serum creatinine and uMCP1. AIN-diagnosed cancer patients also had higher uMCP1 levels than ATN-diagnosed patients, confirming our previous findings. Mice with AIN exhibited interstitial PD-1 staining and stronger glomerular PD-1 expression, especially with combination treatment. Conversely, human AIN patients only showed interstitial PD-1 positivity. CONCLUSIONS: Only mice receiving cisplatin and anti-PDL1 concomitantly developed AIN, accompanied with a more severe kidney injury. AIN induced by this drug combination was linked to elevated uMCP1, consistently with human AIN, suggesting that uMCP1 can be potentially used as an AIN biomarker.
Assuntos
Quimiocina CCL2 , Cisplatino , Inibidores de Checkpoint Imunológico , Camundongos Endogâmicos C57BL , Nefrite Intersticial , Receptor de Morte Celular Programada 1 , Animais , Inibidores de Checkpoint Imunológico/efeitos adversos , Inibidores de Checkpoint Imunológico/farmacologia , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Nefrite Intersticial/urina , Nefrite Intersticial/patologia , Nefrite Intersticial/induzido quimicamente , Quimiocina CCL2/urina , Quimiocina CCL2/metabolismo , Cisplatino/efeitos adversos , Humanos , Masculino , Feminino , Glomérulos Renais/patologia , Glomérulos Renais/efeitos dos fármacos , Antígeno B7-H1/metabolismo , Camundongos , Pessoa de Meia-Idade , Idoso , Doença AgudaRESUMO
The Oxford histopathologic classification (MEST-C: scores for lesions indicating active glomerular inflammation, mesangial [M] and endocapillary [E] hypercellularity as well as cellular or fibrocellular crescents [C], and for segmental glomerulosclerosis [S] and interstitial fibrosis and/or tubular atrophy [T]) is useful in helping assess prognosis in patients with IgA nephropathy. Elements of this classification indicative of active glomerular inflammation, endocapillary hypercellularity and crescents, also have been found to be responsive to immunosuppressive therapy, potentially including newer agents specifically targeting mediators of such inflammation. In this issue of Kidney International, Bellur and coworkers identify histopathologic subtypes of segmental glomerulosclerosis in IgA nephropathy showing podocyte injury that also behave like active lesions, including showing improved outcomes with immunosuppression. This podocyte injury, identifiable only by kidney biopsy, may represent a potential therapeutic target in some patients with IgA nephropathy.
Assuntos
Glomerulonefrite por IGA , Podócitos , Glomerulonefrite por IGA/patologia , Glomerulonefrite por IGA/tratamento farmacológico , Glomerulonefrite por IGA/imunologia , Humanos , Podócitos/patologia , Podócitos/imunologia , Podócitos/efeitos dos fármacos , Biópsia , Imunossupressores/uso terapêutico , Glomerulosclerose Segmentar e Focal/patologia , Glomerulosclerose Segmentar e Focal/imunologia , Glomerulosclerose Segmentar e Focal/tratamento farmacológico , Glomérulos Renais/patologia , Glomérulos Renais/imunologia , Glomérulos Renais/efeitos dos fármacos , PrognósticoRESUMO
Diabetic nephropathy (DN), as a complication of diabetes, is a substantial healthcare challenge owing to the high risk of morbidity and mortality involved. Although significant progress has been made in understanding the pathogenesis of DN, more efficient models are required to develop new therapeutics. Here, we created a DN model in zebrafish by crossing diabetic Tg(acta1:dnIGF1R-EGFP) and proteinuria-tracing Tg(l-fabp::VDBP-GFP) lines, named zMIR/VDBP. Overfed adult zMIR/VDBP fish developed severe hyperglycemia and proteinuria, which were not observed in wild-type zebrafish. Renal histopathology revealed human DN-like characteristics, such as glomerular basement membrane thickening, foot process effacement and glomerular sclerosis. Glomerular dysfunction was restored upon calorie restriction. RNA sequencing analysis demonstrated that DN zebrafish kidneys exhibited transcriptional patterns similar to those seen in human DN pathogenesis. Notably, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was activated, a phenomenon observed in the early phase of human DN. In addition, metformin improved hyperglycemia and proteinuria in DN zebrafish by modulating Akt phosphorylation. Our results indicate that zMIR/VDBP fish are suitable for elucidating the mechanisms underlying human DN and could be a powerful tool for therapeutic discovery.
Assuntos
Nefropatias Diabéticas , Modelos Animais de Doenças , Hiperglicemia , Proteinúria , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Peixe-Zebra , Animais , Hiperglicemia/complicações , Hiperglicemia/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Humanos , Fosforilação/efeitos dos fármacos , Animais Geneticamente Modificados , Metformina/farmacologia , Metformina/uso terapêutico , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Rim/patologia , Rim/efeitos dos fármacos , Rim/metabolismo , Glomérulos Renais/patologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Ativação Enzimática/efeitos dos fármacosRESUMO
Xanthine oxidoreductase (XOR) contributes to reactive oxygen species production. We investigated the cytoprotective mechanisms of XOR inhibition against high glucose (HG)-induced glomerular endothelial injury, which involves activation of the AMP-activated protein kinase (AMPK). Human glomerular endothelial cells (GECs) exposed to HG were subjected to febuxostat treatment for 48 h and the expressions of AMPK and its associated signaling pathways were evaluated. HG-treated GECs were increased xanthine oxidase/xanthine dehydrogenase levels and decreased intracellular AMP/ATP ratio, and these effects were reversed by febuxostat treatment. Febuxostat enhanced the phosphorylation of AMPK, the activation of peroxisome proliferator-activated receptor (PPAR)-gamma coactivator (PGC)-1α and PPAR-α and suppressed the phosphorylation of forkhead box O (FoxO)3a in HG-treated GECs. Febuxostat also decreased nicotinamide adenine dinucleotide phosphate oxidase (Nox)1, Nox2, and Nox4 expressions; enhanced superoxide dismutase activity; and decreased malondialdehyde levels in HG-treated GECs. The knockdown of AMPK inhibited PGC-1α-FoxO3a signaling and negated the antioxidant effects of febuxostat in HG-treated GECs. Despite febuxostat administration, the knockdown of hypoxanthine phosphoribosyl transferase 1 (HPRT1) also inhibited AMPK-PGC-1α-FoxO3a in HG-treated GECs. XOR inhibition alleviates oxidative stress by activating AMPK-PGC-1α-FoxO3a signaling through the HPRT1-dependent purine salvage pathway in GECs exposed to HG conditions.
Assuntos
Proteínas Quinases Ativadas por AMP , Injúria Renal Aguda , Células Endoteliais , Glucose , Purinas , Xantina Desidrogenase , Humanos , Proteínas Quinases Ativadas por AMP/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Febuxostat/farmacologia , Glucose/metabolismo , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Glomérulos Renais/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Purinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Xantina Desidrogenase/antagonistas & inibidores , Xantina Desidrogenase/metabolismo , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/metabolismoRESUMO
The mechanisms responsible for glomerular hemodynamic regulation with sodium-glucose co-transporter 2 (SGLT2) inhibitors in kidney disease due to type 2 diabetes remain unclear. Therefore, we investigated changes in glomerular hemodynamic function using an animal model of type 2 diabetes, treated with an SGLT2 inhibitor alone or in combination with a renin-angiotensin-aldosterone system inhibitor using male Zucker lean (ZL) and Zucker diabetic fatty (ZDF) rats. Afferent and efferent arteriolar diameter and single-nephron glomerular filtration rate (SNGFR) were evaluated in ZDF rats measured at 0, 30, 60, 90, and 120 minutes after the administration of a SGLT2 inhibitor (luseogliflozin). Additionally, we assessed these changes under the administration of the adenosine A1 receptor (A1aR) antagonist (8-cyclopentyl-1,3-dipropylxanthine), along with coadministration of luseogliflozin and an angiotensin II receptor blocker (ARB), telmisartan. ZDF rats had significantly increased SNGFR, and afferent and efferent arteriolar diameters compared to ZL rats, indicating glomerular hyperfiltration. Administration of luseogliflozin significantly reduced afferent vasodilatation and glomerular hyperfiltration, with no impact on efferent arteriolar diameter. Urinary adenosine levels were increased significantly in the SGLT2 inhibitor group compared to the vehicle group. A1aR antagonism blocked the effect of luseogliflozin on kidney function. Co-administration of the SGLT2 inhibitor and ARB decreased the abnormal expansion of glomerular afferent arterioles, whereas the efferent arteriolar diameter was not affected. Thus, regulation of afferent arteriolar vascular tone via the A1aR pathway is associated with glomerular hyperfiltration in type 2 diabetic kidney disease.
Assuntos
Diabetes Mellitus Tipo 2 , Nefropatias Diabéticas , Taxa de Filtração Glomerular , Glomérulos Renais , Ratos Zucker , Inibidores do Transportador 2 de Sódio-Glicose , Animais , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Masculino , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/fisiopatologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/fisiopatologia , Glomérulos Renais/patologia , Glomérulos Renais/irrigação sanguínea , Taxa de Filtração Glomerular/efeitos dos fármacos , Ratos , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/fisiopatologia , Hemodinâmica/efeitos dos fármacos , Xantinas/farmacologia , Arteríolas/efeitos dos fármacos , Arteríolas/fisiopatologia , Antagonistas do Receptor A1 de Adenosina/farmacologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Transportador 2 de Glucose-Sódio/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Sorbitol/análogos & derivadosRESUMO
Adiponectin has vascular anti-inflammatory and protective effects. Although adiponectin protects against the development of albuminuria, historically, the focus has been on podocyte protection within the glomerular filtration barrier (GFB). The first barrier to albumin in the GFB is the endothelial glycocalyx (eGlx), a surface gel-like barrier covering glomerular endothelial cells (GEnCs). In diabetes, eGlx dysfunction occurs before podocyte damage; hence, we hypothesized that adiponectin could protect from eGlx damage to prevent early vascular damage in diabetic kidney disease (DKD). Globular adiponectin (gAd) activated AMPK signaling in human GEnCs through AdipoR1. It significantly reduced eGlx shedding and the tumor necrosis factor-α (TNF-α)-mediated increase in syndecan-4 (SDC4) and MMP2 mRNA expression in GEnCs in vitro. It protected against increased TNF-α mRNA expression in glomeruli isolated from db/db mice and against expression of genes associated with glycocalyx shedding (namely, SDC4, MMP2, and MMP9). In addition, gAd protected against increased glomerular albumin permeability (Ps'alb) in glomeruli isolated from db/db mice when administered intraperitoneally and when applied directly to glomeruli (ex vivo). Ps'alb was inversely correlated with eGlx depth in vivo. In summary, adiponectin restored eGlx depth, which was correlated with improved glomerular barrier function, in diabetes.
Assuntos
Adiponectina , Diabetes Mellitus Tipo 2 , Glicocálix , Glomérulos Renais , Animais , Glicocálix/metabolismo , Glicocálix/efeitos dos fármacos , Adiponectina/metabolismo , Adiponectina/genética , Camundongos , Diabetes Mellitus Tipo 2/metabolismo , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Glomérulos Renais/efeitos dos fármacos , Humanos , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Masculino , Barreira de Filtração Glomerular/metabolismo , Barreira de Filtração Glomerular/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Sindecana-4/metabolismo , Sindecana-4/genética , Modelos Animais de Doenças , Camundongos Endogâmicos C57BLRESUMO
IgA nephropathy (IgAN) is characterized by glomerular deposition of immune complexes (ICs) consisting of IgA1 with O-glycans deficient in galactose (Gd-IgA1) and Gd-IgA1-specific IgG autoantibodies. These ICs induce kidney injury, and in the absence of disease-specific therapy, up to 40% of patients with IgAN progress to kidney failure. IgA1 with its clustered O-glycans is unique to humans, which hampered development of small-animal models of IgAN. Here, we used a model wherein engineered ICs (EICs) formed from human Gd-IgA1 and recombinant human IgG autoantibody are injected into nude mice to induce glomerular injury mimicking human IgAN. In this model, we assessed the protective effects of sparsentan, a single-molecule dual endothelin angiotensin receptor antagonist (DEARA) versus vehicle on EIC-induced glomerular proliferation and dysregulation of gene expression in the kidney. Oral administration of sparsentan (60 or 120 mg/kg daily) to mice intravenously injected with EIC attenuated the EIC-induced glomerular hypercellularity. Furthermore, analysis of changes in the whole kidney transcriptome revealed that key inflammatory and proliferative biological genes and pathways that are upregulated in this EIC model of IgAN were markedly reduced by sparsentan, including complement genes, integrin components, members of the mitogen-activated protein kinase family, and Fc receptor elements. Partial overlap between mouse and human differentially expressed genes in IgAN further supported the translational aspect of the immune and inflammatory components from our transcriptional findings. In conclusion, our data indicate that in the mouse model of IgAN, sparsentan targets immune and inflammatory processes leading to protection from mesangial hypercellularity.NEW & NOTEWORTHY The mechanisms by which deposited IgA1 immune complexes cause kidney injury during early phases of IgA nephropathy are poorly understood. We used an animal model we recently developed that involves IgA1-IgG immune complex injections and determined pathways related to the induced mesangioproliferative changes. Treatment with sparsentan, a dual inhibitor of endothelin type A and angiotensin II type 1 receptors, ameliorated the induced mesangioproliferative changes and the associated alterations in the expression of inflammatory genes and networks.
Assuntos
Complexo Antígeno-Anticorpo , Modelos Animais de Doenças , Glomerulonefrite por IGA , Imunoglobulina A , Imunoglobulina G , Glomérulos Renais , Animais , Glomerulonefrite por IGA/imunologia , Glomerulonefrite por IGA/tratamento farmacológico , Glomerulonefrite por IGA/genética , Glomerulonefrite por IGA/patologia , Glomerulonefrite por IGA/metabolismo , Imunoglobulina A/metabolismo , Imunoglobulina A/imunologia , Glomérulos Renais/patologia , Glomérulos Renais/metabolismo , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/imunologia , Complexo Antígeno-Anticorpo/metabolismo , Redes Reguladoras de Genes , Camundongos Nus , Humanos , Camundongos , Proliferação de Células/efeitos dos fármacosRESUMO
Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce the risk for kidney failure and are a key component of guideline-directed therapy for CKD. While SGLT2 inhibitors' ability to activate tubuloglomerular feedback and reduce hyperfiltration-mediated kidney injury is considered to be the central mechanism for kidney protection, recent data from experimental studies raise questions on the primacy of this mechanism. This review examines SGLT2 inhibitors' role in tubuloglomerular feedback and summarizes emerging evidence on following of SGLT2 inhibitors' other putative mechanisms for kidney protection: optimization of kidney's energy substrate utilization and delivery, regulation of autophagy and maintenance of cellular homeostasis, attenuation of sympathetic hyperactivity, and improvement in vascular health and microvascular function. It is imperative to examine the effect of SGLT2 inhibition on these different physiologic processes to help our understanding of mechanisms underpinning kidney protection with this important class of drugs.
Assuntos
Inibidores do Transportador 2 de Sódio-Glicose , Humanos , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Animais , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/patologia , Glomérulos Renais/irrigação sanguínea , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/prevenção & controle , Insuficiência Renal Crônica/fisiopatologia , Rim/efeitos dos fármacos , Rim/metabolismo , Autofagia/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos dos fármacosRESUMO
Cadmium (Cd) is one of the most polluting heavy metal in the environment. Cd exposure has been elucidated to cause dysfunction of the glomerular filtration barrier (GFB). However, the underlying mechanism remains unclear. C57BL/6J male mice were administered with 2.28 mg/kg cadmium chloride (CdCl2) dissolved in distilled water by oral gavage for 14 days. The expression of SDC4 in the kidney tissues was detected. Human renal glomerular endothelial cells (HRGECs) were exposed to varying concentrations of CdCl2 for 24 h. The mRNA levels of SDC4, along with matrix metalloproteinase (MMP)-2 and 9, were analyzed by quantitative PCR. Additionally, the protein expression levels of SDC4, MMP-2/9, and both total and phosphorylated forms of Smad2/3 (P-Smad2/3) were detected by western blot. The extravasation rate of fluorescein isothiocyanate-dextran through the Transwell was used to evaluate the permeability of HRGECs. SB431542 was used as an inhibitor of transforming growth factor (TGF)-ß signaling pathway to further investigate the role of TGF-ß. Cd reduced SDC4 expression in both mouse kidney tissues and HRGECs. In addition, Cd exposure increased permeability and upregulated P-Smad2/3 levels in HRGECs. SB431542 treatment inhibited the phosphorylation of Smad2/3, Cd-induced SDC4 downregulation, and hyperpermeability. MMP-2/9 levels increased by Cd exposure was also blocked by SB431542, demonstrating the involvement of TGF-ß/Smad pathway in low-dose Cd-induced SDC4 reduction in HRGECs. Given that SDC4 is an essential component of glycocalyx, protection or repair of endothelial glycocalyx is a potential strategy for preventing or treating kidney diseases associated with environmental Cd exposure.
Assuntos
Cádmio , Células Endoteliais , Glicocálix , Glomérulos Renais , Sindecana-4 , Animais , Humanos , Masculino , Camundongos , Cádmio/toxicidade , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Glicocálix/efeitos dos fármacos , Glicocálix/metabolismo , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Sindecana-4/metabolismo , Sindecana-4/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Some chemoattractants and leukocytes such as M1 and M2 macrophages are known to be involved in the development of glomerulosclerosis during diabetic nephropathy (DN). In the course of diabetes, an altered and defective cellular metabolism leads to the increase in adenosine levels, and thus to changes in the polarity (M1/M2) of macrophages. MRS1754, a selective antagonist of the A2B adenosine receptor (A2BAR), attenuated glomerulosclerosis and decreased macrophage-myofibroblast transition in DN rats. Therefore, we aimed to investigate the effect of MRS1754 on the glomerular expression/secretion of chemoattractants, the intraglomerular infiltration of leukocytes, and macrophage polarity in DN rats. Kidneys/glomeruli of non-diabetic, DN, and MRS1754-treated DN rats were processed for transcriptomic analysis, immunohistopathology, ELISA, and in vitro macrophage migration assays. The transcriptomic analysis identified an upregulation of transcripts and pathways related to the immune system in the glomeruli of DN rats, which was attenuated using MRS1754. The antagonism of the A2BAR decreased glomerular expression/secretion of chemoattractants (CCL2, CCL3, CCL6, and CCL21), the infiltration of macrophages, and their polarization to M2 in DN rats. The in vitro macrophages migration induced by conditioned-medium of DN glomeruli was significantly decreased using neutralizing antibodies against CCL2, CCL3, and CCL21. We concluded that the pharmacological blockade of the A2BAR decreases the transcriptional expression of genes/pathways related to the immune response, protein expression/secretion of chemoattractants, as well as the infiltration of macrophages and their polarization toward the M2 phenotype in the glomeruli of DN rats, suggesting a new mechanism implicated in the antifibrotic effect of MRS1754.
Assuntos
Acetamidas , Antagonistas do Receptor A2 de Adenosina , Polaridade Celular , Fatores Quimiotáticos , Nefropatias Diabéticas , Glomérulos Renais , Macrófagos , Purinas , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/imunologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Fatores Quimiotáticos/antagonistas & inibidores , Fatores Quimiotáticos/genética , Fatores Quimiotáticos/metabolismo , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Antagonistas do Receptor A2 de Adenosina/farmacologia , Receptor A2B de Adenosina , Acetamidas/farmacologia , Purinas/farmacologia , Animais , Ratos , Movimento Celular/efeitos dos fármacos , Masculino , Ratos Sprague-Dawley , Transcrição Gênica/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Imunidade/efeitos dos fármacos , Imunidade/genéticaRESUMO
OBJECTIVE: To investigate the role of microRNA-155-5p on apoptosis and inflammatory response in human renal glomerular endothelial cells (HRGEC) cultured with high glucose. METHODS: The primary HRGEC were mainly studied, light microscopy was used to detect changes in cell morphology. Quantitative Real Time-Polymerase Chain Reaction, Western Blot, immunofluorescence were aimed to observe the mRNA and protein expression levels of target gene ETS-1, downstream factors VCAM-1, MCP-1 and cleaved caspase-3 in each group after high glucose treatment as well as transfection with miR-155 mimics or inhibitor. RESULTS: The expression of inflammatory factors and apoptosis of HRGEC cells increased under high glucose treatment. Compared with normal-glucose treatment, the expression of microRNA-155 markedly increased in HRGECs treated with high-glucose, as well as the mRNA and protein levels of ETS-1, VCAM-1, MCP-1 and cleaved caspase-3. Overexpression of microRNA-155 remarkably downregulated mRNA and protein levels of ETS-1, VCAM-1, MCP-1 and cleaved caspase-3, whereas miRNA-155 knockdown upregulated their levels. In addition, HRGEC cells were transfected with miR-155 mimics and ETS-1 siRNA with high glucose stimulation. The expression of ETS-1 was positively correlated with the expression of downstream factors VCAM-1 and MCP-1. These results suggest that ETS-1 can mediate endothelial cell inflammation by regulating VCAM-1 and MCP-1. CONCLUSION: MiR-155 can negatively regulate the expression of target gene ETS-1 and its downstream factors VCAM-1, MCP-1 and cleaved caspase-3, thus mediating the inflammatory response and apoptosis of HRGEC.
Assuntos
Células Endoteliais , MicroRNAs , Apoptose/genética , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Glucose/administração & dosagem , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Diabetic kidney disease (DKD) is one of the microvascular complications of diabetes mellitus and a major cause of end-stage renal disease with limited treatment options. Wogonin is a flavonoid derived from the root of Scutellaria baicalensis Georgi, which has shown a potent renoprotective effect. But the mechanisms of action in DKD are not fully elucidated. In this study, we investigated the effects of wogonin on glomerular podocytes in DKD using mouse podocyte clone 5 (MPC5) cells and diabetic mice model. MPC5 cells were treated with high glucose (30 mM). We showed that wogonin (4, 8, 16 µM) dose-dependently alleviated high glucose (HG)-induced MPC5 cell damage, accompanied by increased expression of WT-1, nephrin, and podocin proteins, and decreased expression of TNF-α, MCP-1, IL-1ß as well as phosphorylated p65. Furthermore, wogonin treatment significantly inhibited HG-induced apoptosis in MPC5 cells. Wogonin reversed HG-suppressed autophagy in MPC5 cells, evidenced by increased ATG7, LC3-II, and Beclin-1 protein, and decreased p62 protein. We demonstrated that wogonin directly bound to Bcl-2 in MPC5 cells. In HG-treated MPC5 cells, knockdown of Bcl-2 abolished the beneficial effects of wogonin, whereas overexpression of Bcl-2 mimicked the protective effects of wogonin. Interestingly, we found that the expression of Bcl-2 was significantly decreased in biopsy renal tissue of diabetic nephropathy patients. In vivo experiments were conducted in STZ-induced diabetic mice, which were administered wogonin (10, 20, 40 mg · kg-1 · d-1, i.g.) every other day for 12 weeks. We showed that wogonin administration significantly alleviated albuminuria, histopathological lesions, and p65 NF-κB-mediated renal inflammatory response. Wogonin administration dose-dependently inhibited podocyte apoptosis and promoted podocyte autophagy in STZ-induced diabetic mice. This study for the first time demonstrates a novel action of wogonin in mitigating glomerulopathy and podocytes injury by regulating Bcl-2-mediated crosstalk between autophagy and apoptosis. Wogonin may be a potential therapeutic drug against DKD.
Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Flavanonas/farmacologia , Glomérulos Renais/efeitos dos fármacos , Podócitos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/administração & dosagem , Flavanonas/administração & dosagem , Injeções Intraperitoneais , Glomérulos Renais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Podócitos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Accidental hypothermia (AH) sometimes leads to coagulation disorder, especially in severe AH. We previously demonstrated that intrasplenic platelet activation caused aberrant hemostasis and thrombus formation after rewarming in a murine AH model. However, no study has focused on the appropriate management of platelets causing coagulation activation after rewarming of AH. We investigated whether or not recombinant soluble thrombomodulin (rTM) can suppress thrombosis formation after rewarming using a rat AH model. METHODS: Wistar rats were exposed to an ambient temperature of -20 °C under general anesthesia until their rectal temperature decreased to 26 °C. The Hypo group rats (n = 5) were immediately euthanized, while the Hypo/Re group (n = 5) and rTM group rats (n = 5), which were administered rTM (1 mg/kg) via the tail vein, were rewarmed until the rectal temperature returned to 34 °C and then euthanized 6 h later. Tissue and blood samples were collected from all rats for histopathological and coagulation analyses at euthanasia. RESULTS: There was no significant change in the D-dimer level in the Hypo group rats, while the D-dimer level was significantly elevated at 6 h after rewarming in the Hypo/Re group rats (P = 0.015), and histopathology detected both fibrin and platelets in the renal glomerulus. However, the rTM group rats did not show any elevation of the D-dimer levels at 6 h after rewarming, and no fibrin was noted on histopathology. CONCLUSIONS: rTM may be useful as an appropriate anticoagulant in cases of aberrant hemostasis after rewarming of AH.
Assuntos
Anticoagulantes/farmacologia , Plaquetas/efeitos dos fármacos , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Hipotermia/complicações , Trombomodulina/administração & dosagem , Trombose/prevenção & controle , Animais , Biomarcadores/metabolismo , Plaquetas/metabolismo , Plaquetas/patologia , Modelos Animais de Doenças , Fibrina/química , Fibrina/metabolismo , Hipotermia/sangue , Hipotermia/fisiopatologia , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Ativação Plaquetária/efeitos dos fármacos , Ratos , Ratos Wistar , Proteínas Recombinantes/farmacologia , Reaquecimento/efeitos adversos , Solubilidade , Baço/irrigação sanguínea , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/patologia , Trombose/sangue , Trombose/etiologia , Trombose/fisiopatologiaRESUMO
Novel strategies for the prevention and treatment of sepsis-associated acute kidney injury and its long-term outcomes have been required and remain a challenge in critical care medicine. Therapeutic strategies using lipid mediators, such as aspirin-triggered resolvin D1 (ATRvD1), can contribute to the resolution of acute and chronic inflammation. In this study, we examined the potential effect of ATRvD1 on long-term kidney dysfunction after severe sepsis. Fifteen days after cecal ligation and puncture (CLP), sepsis-surviving BALB/c mice were subjected to a tubulointerstitial injury through intraperitoneal injections of bovine serum albumin (BSA) for 7 days, called the subclinical acute kidney injury (subAKI) animal model. ATRvD1 treatment was performed right before BSA injections. On day 22 after CLP, the urinary protein/creatinine ratio (UPC), histologic parameters, fibrosis, cellular infiltration, apoptosis, inflammatory markers levels, and mRNA expression were determined. ATRvD1 treatment mitigated tubulointerstitial injury by reducing proteinuria excretion, the UPC ratio, the glomerular cell number, and extracellular matrix deposition. Pro-fibrotic markers, such as transforming growth factor ß (TGFß), type 3 collagen, and metalloproteinase (MMP)-3 and -9 were reduced after ATRvD1 administration. Post-septic mice treated with ATRvD1 were protected from the recruitment of IBA1+ cells. The interleukin-1ß (IL-1ß) levels were increased in the subAKI animal model, being attenuated by ATRvD1. Tumor necrosis factor-α (TNF-α), IL-10, and IL-4 mRNA expression were increased in the kidney of BSA-challenged post-septic mice, and it was also reduced after ATRvD1. These results suggest that ATRvD1 protects the kidney against a second insult such as BSA-induced tubulointerstitial injury and fibrosis by suppressing inflammatory and pro-fibrotic mediators in renal dysfunction after sepsis.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Aspirina/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Glomérulos Renais/efeitos dos fármacos , Sepse/tratamento farmacológico , Injúria Renal Aguda/induzido quimicamente , Albuminas/farmacologia , Animais , Biomarcadores/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Testes de Função Renal/métodos , Glomérulos Renais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteinúria/induzido quimicamente , Proteinúria/tratamento farmacológico , Proteinúria/metabolismo , RNA Mensageiro/metabolismo , Sepse/metabolismoRESUMO
Hemolytic Uremic Syndrome (HUS) associated with Shiga-toxigenic Escherichia coli (STEC) infections is the principal cause of acute renal injury in pediatric age groups. Shiga toxin type 2 (Stx2) has in vitro cytotoxic effects on kidney cells, including human glomerular endothelial (HGEC) and Vero cells. Neither a licensed vaccine nor effective therapy for HUS is available for humans. Recombinant antibodies against Stx2, produced in bacteria, appeared as the utmost tool to prevent HUS. Therefore, in this work, a recombinant FabF8:Stx2 was selected from a human Fab antibody library by phage display, characterized, and analyzed for its ability to neutralize the Stx activity from different STEC-Stx2 and Stx1/Stx2 producing strains in a gold standard Vero cell assay, and the Stx2 cytotoxic effects on primary cultures of HGEC. This recombinant Fab showed a dissociation constant of 13.8 nM and a half maximum effective concentration (EC50) of 160 ng/mL to Stx2. Additionally, FabF8:Stx2 neutralized, in different percentages, the cytotoxic effects of Stx2 and Stx1/2 from different STEC strains on Vero cells. Moreover, it significantly prevented the deleterious effects of Stx2 in a dose-dependent manner (up to 83%) in HGEC and protected this cell up to 90% from apoptosis and necrosis. Therefore, this novel and simple anti-Stx2 biomolecule will allow further investigation as a new therapeutic option that could improve STEC and HUS patient outcomes.
Assuntos
Anticorpos Monoclonais/farmacologia , Síndrome Hemolítico-Urêmica/prevenção & controle , Fragmentos Fab das Imunoglobulinas/imunologia , Toxina Shiga II/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Apoptose/efeitos dos fármacos , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Humanos , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Glomérulos Renais/citologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/patologia , Proteínas Recombinantes , Toxina Shiga I/imunologia , Toxina Shiga I/toxicidade , Toxina Shiga II/toxicidade , Escherichia coli Shiga Toxigênica/imunologia , Células VeroRESUMO
Increased expression and activity of the Ca2+ channel transient receptor potential channel 6 (TRPC6) is associated with focal segmental glomerulosclerosis, but therapeutic strategies to target TRPC6 are currently lacking. Nitric oxide (NO) is crucial for normal glomerular function and plays a protective role in preventing glomerular diseases. We investigated if NO prevents podocyte injury by inhibiting injurious TRPC6-mediated signaling in a soluble guanylate cyclase (sGC)-dependent manner and studied the therapeutic potential of the sGC stimulator Riociguat. Experiments were performed using human glomerular endothelial cells and podocytes. Podocyte injury was induced by Adriamycin incubation for 24 h, with or without the NO-donor S-Nitroso-N-acetyl-DL-penicillamine (SNAP), the sGC stimulator Riociguat or the TRPC6 inhibitor Larixyl Acetate (LA). NO and Riociguat stimulated cGMP synthesis in podocytes, decreased Adriamycin-induced TRPC6 expression, inhibited the Adriamycin-induced TRPC6-mediated Ca2+ influx and reduced podocyte injury. The protective effects of Riociguat and NO were blocked when sGC activity was inhibited with 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or when TRPC6 activity was inhibited by LA. Our data demonstrate a glomerular (e)NOS-NO-sGC-cGMP-TRPC6 pathway that prevents podocyte injury, which can be translated to future clinical use by, e.g., repurposing the market-approved drug Riociguat.
Assuntos
Guanilato Ciclase/genética , Óxido Nítrico/genética , Podócitos/efeitos dos fármacos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Canal de Cátion TRPC6/genética , Animais , Sinalização do Cálcio/efeitos dos fármacos , GMP Cíclico/genética , Reposicionamento de Medicamentos , Células Endoteliais/efeitos dos fármacos , Humanos , Nefropatias/tratamento farmacológico , Nefropatias/genética , Nefropatias/patologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/patologia , Camundongos , Comunicação Parácrina/efeitos dos fármacos , Podócitos/patologiaRESUMO
Chronic kidney disease (CKD) is characterized by the gradual loss of renal function and is a major public health concern. Risk factors for CKD include hypertension and proteinuria, both of which are associated with endoplasmic reticulum (ER) stress. ER stress-induced TDAG51 protein expression is increased at an early time point in mice with CKD. Based on these findings, wild-type and TDAG51 knock-out (TDKO) mice were used in an angiotensin II/deoxycorticosterone acetate/salt model of CKD. Both wild-type and TDKO mice developed hypertension, increased proteinuria and albuminuria, glomerular injury, and tubular damage. However, TDKO mice were protected from apoptosis and renal interstitial fibrosis. Human proximal tubular cells were used to demonstrate that TDAG51 expression induces apoptosis through a CHOP-dependent mechanism. Further, a mouse model of intrinsic acute kidney injury demonstrated that CHOP is required for ER stress-mediated apoptosis. Renal fibroblasts were used to demonstrate that TGF-ß induces collagen production through an IRE1-dependent mechanism; cells treated with a TGF-ß receptor 1 inhibitor prevented XBP1 splicing, a downstream consequence of IRE1 activation. Interestingly, TDKO mice express significantly less TGF-ß receptor 1, thus, preventing TGF-ß-mediated XBP1 splicing. In conclusion, TDAG51 induces apoptosis in the kidney through a CHOP-dependent mechanism, while contributing to renal interstitial fibrosis through a TGF-ß-IRE1-XBP1 pathway.
Assuntos
Rim/patologia , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Fatores de Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Humanos , Himecromona/análogos & derivados , Himecromona/farmacologia , Rim/efeitos dos fármacos , Rim/fisiopatologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/patologia , Glomérulos Renais/fisiopatologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , Túbulos Renais/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Ratos , Insuficiência Renal Crônica/fisiopatologia , Fatores de Risco , Fator de Transcrição CHOP/metabolismo , Tunicamicina/farmacologia , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
Vascular endothelial cells are covered with glycocalyx comprising heparan sulfate, hyaluronan, chondroitin sulfate, and associated proteins. Glomerular endothelial glycocalyx is involved in protecting against induction of proteinuria and structural damage, but the specific components in glycocalyx that represent therapeutic targets remain unclear. Anti-vascular endothelial growth factor (VEGF) therapy is associated with an increased risk of glomerular endothelial injury. This study investigated whether hyaluronan could provide a therapeutic target to protect against proteinuria. We conducted ex vivo and in vivo experiments to explore the effects of degrading glomerular hyaluronan by administering hyaluronidase and of supplementation with hyaluronan. We investigated hyaluronan expression using biotin-labeled hyaluronan-binding protein (HABP) in human kidney specimens or serum hyaluronan in endothelial injuries under inhibition of VEGF signaling. We directly demonstrated hyaluronan in glomerular endothelial layers using HABP staining. Ex vivo and in vivo experiments showed the development of proteinuria after digestion of hyaluronan in glomerular capillaries. Supplementation with hyaluronan after hyaluronidase treatment suppressed proteinuria. Mice in the in vivo study developed albuminuria after intraperitoneal injection of hyaluronidase with decreased glomerular hyaluronan and increased serum hyaluronan. In human kidneys with endothelial cell dysfunction and proteinuria due to inhibition of VEGF, glomerular expression of hyaluronan was reduced even in normal-appearing glomeruli. Serum hyaluronan levels were elevated in patients with pre-eclampsia with VEGF signaling inhibition. Our data suggest that hyaluronan itself plays crucial roles in preventing proteinuria and preserving the integrity of endothelial cells. Hyaluronan could provide a therapeutic target for preventing glomerular endothelial glycocalyx damage, including VEGF signaling inhibition.