RESUMO
The lack of standardization in antibody validation remains a major contributor to irreproducibility of human research. To address this, we have applied a standardized approach to validate a panel of antibodies to identify 18 major cell types and 5 extracellular matrix compartments in the human kidney by immunofluorescence (IF) microscopy. We have used these to generate an organ mapping antibody panel for two-dimensional (2-D) and three-dimensional (3-D) cyclical IF (CyCIF) to provide a more detailed method for evaluating tissue segmentation and volumes using a larger panel of markers than would normally be possible using standard fluorescence microscopy. CyCIF also makes it possible to perform multiplexed IF microscopy of whole slide images, which is a distinct advantage over other multiplexed imaging technologies that are applicable to limited fields of view. This enables a broader view of cell distributions across larger anatomical regions, allowing a better chance to capture localized regions of dysfunction in diseased tissues. These methods are broadly accessible to any laboratory with a fluorescence microscope, enabling spatial cellular phenotyping in normal and disease states. We also provide a detailed solution for image alignment between CyCIF cycles that can be used by investigators to perform these studies without programming experience using open-sourced software. This ability to perform multiplexed imaging without specialized instrumentation or computational skills opens the door to integration with more highly dimensional molecular imaging modalities such as spatial transcriptomics and imaging mass spectrometry, enabling the discovery of molecular markers of specific cell types, and how these are altered in disease.NEW & NOTEWORTHY We describe here validation criteria used to define on organ mapping panel of antibodies that can be used to define 18 cell types and five extracellular matrix compartments using cyclical immunofluorescence (CyCIF) microscopy. As CyCIF does not require specialized instrumentation, and image registration required to assemble CyCIF images can be performed by any laboratory without specialized computational skills, this technology is accessible to any laboratory with access to a fluorescence microscope and digital scanner.
Assuntos
Anticorpos , Rim , Microscopia de Fluorescência , Humanos , Microscopia de Fluorescência/métodos , Rim/imunologia , Rim/metabolismo , Anticorpos/imunologia , Imunofluorescência/métodos , Reprodutibilidade dos Testes , Matriz Extracelular/metabolismo , Matriz Extracelular/imunologia , Imageamento Tridimensional/métodosRESUMO
Acute kidney injury (AKI) is common in surgical and critically ill patients. This study examined whether pretreatment with a novel Toll-like receptor 4 agonist attenuated ischemia-reperfusion injury (IRI)-induced AKI (IRI-AKI). We performed a blinded, randomized-controlled study in mice pretreated with 3-deacyl 6-acyl phosphorylated hexaacyl disaccharide (PHAD), a synthetic Toll-like receptor 4 agonist. Two cohorts of male BALB/c mice received intravenous vehicle or PHAD (2, 20, or 200 µg) at 48 and 24 h before unilateral renal pedicle clamping and simultaneous contralateral nephrectomy. A separate cohort of mice received intravenous vehicle or 200 µg PHAD followed by bilateral IRI-AKI. Mice were monitored for evidence of kidney injury for 3 days postreperfusion. Kidney function was assessed by serum blood urea nitrogen and creatinine measurements. Kidney tubular injury was assessed by semiquantitative analysis of tubular morphology on periodic acid-Schiff (PAS)-stained kidney sections and by kidney mRNA quantification of injury [neutrophil gelatinase-associated lipocalin (Ngal), kidney injury molecule-1 (Kim-1), and heme oxygenase-1 (Ho-1)] and inflammation [interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (Tnf-α)] using quantitative RT-PCR. Immunohistochemistry was used to quantify proximal tubular cell injury and renal macrophages by quantifying the areas stained with Kim-1 and F4/80 antibodies, respectively, and TUNEL staining to detect the apoptotic nuclei. PHAD pretreatment yielded dose-dependent kidney function preservation after unilateral IRI-AKI. Histological injury, apoptosis, Kim-1 staining, and Ngal mRNA were lower in PHAD-treated mice and IL-1ß mRNA was higher in PHAD-treated mice. Similar pretreatment protection was noted with 200 mg PHAD after bilateral IRI-AKI, with significantly reduced Kim-1 immunostaining in the outer medulla of mice treated with PHAD after bilateral IRI-AKI. In conclusion, PHAD pretreatment leads to dose-dependent protection from renal injury after unilateral and bilateral IRI-AKI in mice.NEW & NOTEWORTHY Pretreatment with 3-deacyl 6-acyl phosphorylated hexaacyl disaccharide; a novel synthetic Toll-like receptor 4 agonist, preserves kidney function during ischemia-reperfusion injury-induced acute kidney injury.
Assuntos
Injúria Renal Aguda , Traumatismo por Reperfusão , Receptor 4 Toll-Like , Animais , Masculino , Camundongos , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/patologia , Injúria Renal Aguda/prevenção & controle , Rim/patologia , Lipocalina-2 , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/patologia , RNA Mensageiro , Receptor 4 Toll-Like/agonistasRESUMO
BACKGROUND: Renal ischemia and reperfusion (IR) contribute to perioperative acute kidney injury, and oxygen is a key regulator of this process. We hypothesized that oxygen administration during surgery and renal IR would impact postoperative kidney function and injury in mice. METHODS: Mice were anesthetized, intubated, and mechanically ventilated with a fraction of inspired oxygen (F io2 ) 0.10 (hypoxia), 0.21 (normoxia), 0.60 (moderate hyperoxia), or 1.00 (severe hyperoxia) during 67 minutes of renal IR or sham IR surgery. Additional mice were treated before IR or sham IR surgery with 50 mg/kg tempol, a superoxide scavenger. At 24 hours, mice were sacrificed, and blood and kidney collected. We assessed and compared kidney function and injury across groups by measuring blood urea nitrogen (BUN, primary end point), renal histological injury, renal expression of neutrophil gelatinase-associated lipocalin (NGAL), and renal heme oxygenase 1 ( Ho-1 ), peroxisome proliferator-activated receptor gamma coactivator 1-α ( Pgc1-α ), and glutathione peroxidase 4 ( Gpx-4 ) transcripts, to explore potential mechanisms of any effect of oxygen. RESULTS: Hyperoxia and hypoxia during renal IR surgery decreased renal function and increased kidney injury compared to normoxia. Baseline median (interquartile range) BUN was 22.2 mg/dL (18.4-26.0), and 24 hours after IR surgery, BUN was 17.5 mg/dL (95% confidence interval [CI], 1.3-38.4; P = .034) higher in moderate hyperoxia-treated animals, 51.8 mg/dL (95% CI, 24.9-74.8; P < .001) higher in severe hyperoxia-treated animals, and 64.9 mg/dL (95% CI, 41.2-80.3; P < .001) higher in hypoxia-treated animals compared to animals treated with normoxia ( P < .001, overall effect of hyperoxia). Hyperoxia-induced injury, but not hypoxia-induced injury, was attenuated by pretreatment with tempol. Histological injury scores, renal NGAL staining, and renal transcription of Ho-1 and suppression of Pgc1- α followed the same pattern as BUN, in relation to the effects of oxygen treatment. CONCLUSIONS: In this controlled preclinical study of oxygen treatment during renal IR surgery, hyperoxia and hypoxia impaired renal function, increased renal injury, and impacted expression of genes that affect mitochondrial biogenesis and antioxidant response. These results might have implications for patients during surgery when high concentrations of oxygen are frequently administered, especially in cases involving renal IR.
RESUMO
Acute kidney injury impacts â¼13.3 million individuals and causes â¼1.7 million deaths per year globally. Numerous injury pathways contribute to acute kidney injury, including cell cycle arrest, senescence, inflammation, mitochondrial dysfunction, and endothelial injury and dysfunction, and can lead to chronic inflammation and fibrosis. However, factors enabling productive repair versus nonproductive, persistent injury states remain less understood. The (Re)Building a Kidney (RBK) consortium is a National Institute of Diabetes and Digestive and Kidney Diseases consortium focused on both endogenous kidney repair mechanisms and the generation of new kidney tissue. This short review provides an update on RBK studies of endogenous nephron repair, addressing the following questions: (i) What is productive nephron repair? (ii) What are the cellular sources and drivers of repair? and (iii) How do RBK studies promote development of therapeutics? Also, we provide a guide to RBK's open access data hub for accessing, downloading, and further analyzing data sets.
Assuntos
Injúria Renal Aguda , Rim , Injúria Renal Aguda/patologia , Feminino , Fibrose , Humanos , Inflamação/patologia , Rim/patologia , Masculino , National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) , Regeneração , Estados UnidosRESUMO
Tubular atrophy and fibrosis are pathological changes that determine the prognosis of kidney disease induced by acute kidney injury (AKI). We aimed to evaluate multiple magnetic resonance imaging (MRI) parameters, including pool size ratio (PSR) from quantitative magnetization transfer, relaxation rates, and measures from spin-lock imaging ( R 1 ρ and S ρ ), for assessing the pathological changes associated with AKI-induced kidney disease. Eight-week-old male C57BL/6 J mice first underwent unilateral ischemia reperfusion injury (IRI) induced by reperfusion after 45 min of ischemia. They were imaged using a 7T MRI system 56 days after the injury. Paraffin tissue sections were stained using Masson trichrome and picrosirius red to identify histopathological changes such as tubular atrophy and fibrosis. Histology detected extensive tubular atrophy and moderate fibrosis in the cortex and outer stripe of the outer medulla (CR + OSOM) and more prominent fibrosis in the inner stripe of the outer medulla (ISOM) of IRI kidneys. In the CR + OSOM region, evident decreases in PSR, R 1 , R 2 , R 1 ρ , and S ρ showed in IRI compared with contralateral kidneys, with PSR and S ρ exhibiting the most significant changes. In addition, the exchange parameter S ρ dropped by the largest degree among all the MRI parameters, while R 2 * increased significantly. In the ISOM of IRI kidneys, PSR increased while S ρ kept decreasing. R 2 , R 1 ρ , and R 2 * all increased due to more severe fibrosis in this region. Among MRI measures, PSR and R 1 ρ showed the highest detectability of renal changes no matter whether tubular atrophy or fibrosis dominated. R 2 * and S ρ could be more specific to a single pathological event than other MRI measures because only R 2 * increased and S ρ decreased consistently when either fibrosis or tubular atrophy dominated, and their correlations with fibrosis scores were higher than other MRI measures. Multiparametric MRI may enable a more comprehensive analysis of histopathological changes following AKI.
Assuntos
Injúria Renal Aguda , Imageamento por Ressonância Magnética Multiparamétrica , Traumatismo por Reperfusão , Injúria Renal Aguda/diagnóstico por imagem , Injúria Renal Aguda/etiologia , Animais , Atrofia/complicações , Atrofia/patologia , Fibrose , Isquemia/patologia , Rim/diagnóstico por imagem , Rim/patologia , Imageamento por Ressonância Magnética/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reperfusão/efeitos adversos , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/diagnóstico por imagem , Traumatismo por Reperfusão/patologiaRESUMO
Treating acute kidney injury (AKI) represents an important unmet medical need both in terms of the seriousness of this medical problem and the number of patients. There is also a large untapped market opportunity in treating AKI. Over the years, there has been much effort in search of therapeutics with minimal success. However, over the same time period, new understanding of the underlying pathobiology and molecular mechanisms of kidney injury have undoubtedly helped the search for new therapeutics. Along this line, carbon monoxide (CO) has emerged as a promising therapeutic agent because of its demonstrated cytoprotective, and immunomodulatory effects. CO has also been shown to sensitize cancer, but not normal cells, to chemotherapy. This is particularly important in treating cisplatin-induced AKI, a common clinical problem that develops in patients receiving cisplatin therapies for a number of different solid organ malignancies. This review will examine and make the case that CO be developed into a therapeutic agent against AKI.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Monóxido de Carbono/uso terapêutico , Injúria Renal Aguda/etiologia , Animais , Monóxido de Carbono/administração & dosagem , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , HumanosRESUMO
Matsushita et al. describe a model of acute kidney injury to chronic kidney disease progression in mice surviving cardiac arrest: mice develop severe acute kidney injury that initially recovers but is followed by the onset of impaired renal function on longer-term follow-up. These findings suggest that distinct cardiorenal toxicities and/or injury dynamics are operative in this cardiac arrest model that do not occur in traditional models of acute kidney injury, providing new opportunities for therapeutic and biomarker discovery for an important clinical problem.
Assuntos
Injúria Renal Aguda , Síndrome Cardiorrenal , Parada Cardíaca , Insuficiência Renal Crônica , Animais , Progressão da Doença , Humanos , Inflamação , CamundongosRESUMO
BACKGROUND: The discoidin domain receptor 1 (DDR1) is activated by collagens, upregulated in injured and fibrotic kidneys, and contributes to fibrosis by regulating extracellular matrix production, but how DDR1 controls fibrosis is poorly understood. DDR1 is a receptor tyrosine kinase (RTK). RTKs can translocate to the nucleus via a nuclear localization sequence (NLS) present on the receptor itself or a ligand it is bound to. In the nucleus, RTKs regulate gene expression by binding chromatin directly or by interacting with transcription factors. METHODS: To determine whether DDR1 translocates to the nucleus and whether this event is mediated by collagen-induced DDR1 activation, we generated renal cells expressing wild-type or mutant forms of DDR1 no longer able to bind collagen. Then, we determined the location of the DDR1 upon collagen stimulation. Using both biochemical assays and immunofluorescence, we analyzed the steps involved in DDR1 nuclear translocation. RESULTS: We show that although DDR1 and its natural ligand, collagen, lack an NLS, DDR1 is present in the nucleus of injured human and mouse kidney proximal tubules. We show that DDR1 nuclear translocation requires collagen-mediated receptor activation and interaction of DDR1 with SEC61B, a component of the Sec61 translocon, and nonmuscle myosin IIA and ß-actin. Once in the nucleus, DDR1 binds to chromatin to increase the transcription of collagen IV, a major collagen upregulated in fibrosis. CONCLUSIONS: These findings reveal a novel mechanism whereby activated DDR1 translates to the nucleus to regulate synthesis of profibrotic molecules.
Assuntos
Colágeno Tipo IV/genética , Colágeno Tipo I/metabolismo , Receptor com Domínio Discoidina 1/metabolismo , Túbulos Renais Proximais/metabolismo , Actinas/metabolismo , Injúria Renal Aguda/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Núcleo Celular , Cromatina/metabolismo , Colágeno Tipo I/farmacologia , Colágeno Tipo IV/metabolismo , Receptor com Domínio Discoidina 1/genética , Humanos , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Cadeias Pesadas de Miosina/metabolismo , Sinais de Localização Nuclear , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Canais de Translocação SEC/metabolismo , Transcrição GênicaRESUMO
Acute kidney injury (AKI) is a strong independent predictor of mortality and often results in incomplete recovery of renal function, leading to progressive chronic kidney disease (CKD). Many clinical trials have been conducted on the basis of promising preclinical data, but no therapeutic interventions have been shown to improve long-term outcomes after AKI. This is partly due to the failure of preclinical studies to accurately model clinically relevant injury and long-term outcomes on CKD progression. Here, we evaluated the long-term effects of AKI on CKD progression in three animal models reflecting diverse etiologies of AKI: repeat-dose cisplatin, rhabdomyolysis, and ischemia-reperfusion injury. Using transdermal measurement of glomerular filtration rate as a clinically relevant measure of kidney function and quantification of peritubular capillary density to measure capillary rarefaction, we showed that repeat-dose cisplatin caused capillary rarefaction and decreased renal function in mice without a significant increase in interstitial fibrosis, whereas rhabdomyolysis-induced AKI led to severe interstitial fibrosis, but renal function and peritubular capillary density were preserved. Furthermore, long-term experiments in mice with unilateral ischemia-reperfusion injury showed that restoration of renal function 12 wk after a contralateral nephrectomy was associated with increasing fibrosis, but a reversal of capillary rarefaction was seen at 4 wk. These data demonstrate that clear dissociation between kidney function and fibrosis in these models of AKI to CKD progression and suggest that peritubular capillary rarefaction is more strongly associated with CKD progression than renal fibrosis.
Assuntos
Injúria Renal Aguda/etiologia , Cisplatino/toxicidade , Rarefação Microvascular/patologia , Insuficiência Renal Crônica/patologia , Traumatismo por Reperfusão/complicações , Rabdomiólise/complicações , Animais , Antineoplásicos/toxicidade , Fibrose/etiologia , Rim/efeitos dos fármacos , Rim/patologia , Masculino , Camundongos , Rarefação Microvascular/etiologiaRESUMO
Severe acute kidney injury has a high mortality and is a risk factor for progressive chronic kidney disease. None of the potential therapies that have been identified in preclinical studies have successfully improved clinical outcomes. This failure is partly because animal models rarely reflect the complexity of human disease: most preclinical studies are short term and are commonly performed in healthy, young, male mice. Therapies that are effective in preclinical models that share common clinical features seen in patients with acute kidney injury, including genetic diversity, different sexes, and comorbidities, and evaluate long-term outcomes are more likely to predict success in the clinic. Here, we evaluated susceptibility to chronic kidney disease after ischemia-reperfusion injury with delayed nephrectomy by monitoring long-term functional and histological responses to injury. We defined conditions required to induce long-term postinjury renal dysfunction and fibrosis without increased mortality in a reproducible way and evaluate effect of mouse strains, sexes, and preexisting diabetes on these responses.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/etiologia , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/complicações , Nefropatias Diabéticas/tratamento farmacológico , Modelos Animais de Doenças , Feminino , Fibrose , Testes de Função Renal , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Nefrectomia , Caracteres Sexuais , Especificidade da EspécieRESUMO
Acute kidney injury is a common complication of severe sepsis and contributes to high mortality. The molecular mechanisms of acute kidney injury during sepsis are not fully understood. Because hemoproteins, including myoglobin and hemoglobin, are known to mediate kidney injury during rhabdomyolysis, we hypothesized that cell-free hemoglobin (CFH) would exacerbate acute kidney injury during sepsis. Sepsis was induced in mice by intraperitoneal injection of cecal slurry (CS). To mimic elevated levels of CFH observed during human sepsis, mice also received a retroorbital injection of CFH or dextrose control. Four groups of mice were analyzed: sham treated (sham), CFH alone, CS alone, and CS + CFH. The addition of CFH to CS reduced 48-h survival compared with CS alone (67% vs. 97%, P = 0.001) and increased the severity of illness. After 24 and 48 h, CS + CFH mice had a reduced glomerular filtration rate from baseline, whereas sham, CFH, and CS mice maintained baseline glomerular filtration rate. Biomarkers of acute kidney injury, neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), were markedly elevated in CS+CFH compared with CS (8-fold for NGAL and 2.4-fold for KIM-1, P < 0.002 for each) after 48 h. Histological examination showed a trend toward increased tubular injury in CS + CFH-exposed kidneys compared with CS-exposed kidneys. However, there were similar levels of renal oxidative injury and apoptosis in the CS + CFH group compared with the CS group. Kidney levels of multiple proinflammatory cytokines were similar between CS and CS + CFH groups. Human renal tubule cells (HK-2) exposed to CFH demonstrated increased cytotoxicity. Together, these results show that CFH exacerbates acute kidney injury in a mouse model of experimental sepsis, potentially through increased renal tubular injury.
Assuntos
Injúria Renal Aguda/patologia , Hemoglobinas/toxicidade , Sepse/patologia , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/fisiopatologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sistema Livre de Células , Citocinas/metabolismo , Feminino , Taxa de Filtração Glomerular , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Lipocalina-2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sepse/complicações , Análise de SobrevidaRESUMO
Retinoic acid (RA) has been used therapeutically to reduce injury and fibrosis in models of AKI, but little is known about the regulation of this pathway and what role it has in regulating injury and repair after AKI. In these studies, we show that RA signaling is activated in mouse and zebrafish models of AKI, and that these responses limit the extent of injury and promote normal repair. These effects were mediated through a novel mechanism by which RA signaling coordinated the dynamic equilibrium of inflammatory M1 spectrum versus alternatively activated M2 spectrum macrophages. Our data suggest that locally synthesized RA represses proinflammatory macrophages, thereby reducing macrophage-dependent injury post-AKI, and activates RA signaling in injured tubular epithelium, which in turn promotes alternatively activated M2 spectrum macrophages. Because RA signaling has an essential role in kidney development but is repressed in the adult, these findings provide evidence of an embryonic signaling pathway that is reactivated after AKI and involved in reducing injury and enhancing repair.
Assuntos
Injúria Renal Aguda/etiologia , Macrófagos/fisiologia , Transdução de Sinais , Tretinoína/fisiologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Hereditary pulmonary arterial hypertension (HPAH) is a rare, fatal disease of the pulmonary vasculature. The majority of HPAH patients inherit mutations in the bone morphogenetic protein type 2 receptor gene (BMPR2), but how these promote pulmonary vascular disease is unclear. HPAH patients have features of pulmonary endothelial cell (PEC) dysfunction including increased vascular permeability and perivascular inflammation associated with decreased PEC barrier function. Recently, frameshift mutations in the caveolar structural protein gene Caveolin-1 (CAV-1) were identified in two patients with non-BMPR2-associated HPAH. Because caveolae regulate endothelial function and vascular permeability, we hypothesized that defects in caveolar function might be a common mechanism by which BMPR2 mutations promote pulmonary vascular disease. To explore this, we isolated PECs from mice carrying heterozygous null Bmpr2 mutations (Bmpr2(+/-)) similar to those found in the majority of HPAH patients. We show that Bmpr2(+/-) PECs have increased numbers and intracellular localization of caveolae and caveolar structural proteins CAV-1 and Cavin-1 and that these defects are reversed after blocking endocytosis with dynasore. SRC kinase is also constitutively activated in Bmpr2(+/-) PECs, and localization of CAV-1 to the plasma membrane is restored after treating Bmpr2(+/-) PECs with the SRC kinase inhibitor 3-(4-chlorophenyl)-1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2). Late outgrowth endothelial progenitor cells isolated from HPAH patients show similar increased activation of SRC kinase. Moreover, Bmpr2(+/-) PECs have impaired endothelial barrier function, and barrier function is restored after treatment with PP2. These data suggest that heterozygous null BMPR2 mutations promote SRC-dependent caveolar trafficking defects in PECs and that this may contribute to pulmonary endothelial barrier dysfunction in HPAH patients.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Hipertensão Pulmonar Primária Familiar/genética , Transporte Proteico/genética , Quinases da Família src/genética , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Cavéolas/metabolismo , Cavéolas/patologia , Caveolina 1/genética , Endocitose/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Hipertensão Pulmonar Primária Familiar/fisiopatologia , Mutação da Fase de Leitura , Heterozigoto , Humanos , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Pirimidinas/administração & dosagem , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
The current lack of effective therapeutics for patients with acute kidney injury (AKI) represents an important and unmet medical need. Given the importance of the clinical problem, it is time for us to take a few steps back and reexamine current practices. The focus of this review is to explore the extent to which failure of therapeutic translation from animal studies to human studies stems from deficiencies in the preclinical models of AKI. We will evaluate whether the preclinical models of AKI that are commonly used recapitulate the known pathophysiologies of AKI that are being modeled in humans, focusing on four common scenarios that are studied in clinical therapeutic intervention trials: cardiac surgery-induced AKI; contrast-induced AKI; cisplatin-induced AKI; and sepsis associated AKI. Based on our observations, we have identified a number of common limitations in current preclinical modeling of AKI that could be addressed. In the long term, we suggest that progress in developing better preclinical models of AKI will depend on developing a better understanding of human AKI. To this this end, we suggest that there is a need to develop greater in-depth molecular analyses of kidney biopsy tissues coupled with improved clinical and molecular classification of patients with AKI.
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Injúria Renal Aguda/etiologia , Modelos Animais de Doenças , Pesquisa Translacional Biomédica , Animais , Antineoplásicos/efeitos adversos , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Cisplatino/efeitos adversos , Meios de Contraste/efeitos adversos , Humanos , Sepse/complicaçõesRESUMO
Acute kidney injury (AKI) is a common and independent risk factor for death and chronic kidney disease (CKD). Despite promising preclinical data, there is no evidence that antioxidants reduce the severity of injury, increase recovery, or prevent CKD in patients with AKI. Pyridoxamine (PM) is a structural analog of vitamin B6 that interferes with oxidative macromolecular damage via a number of different mechanisms and is in a phase 3 clinical efficacy trial to delay CKD progression in patients with diabetic kidney disease. Because oxidative stress is implicated as one of the main drivers of renal injury after AKI, the ability of PM to interfere with multiple aspects of oxidative damage may be favorable for AKI treatment. In these studies we therefore evaluated PM treatment in a mouse model of AKI. Pretreatment with PM caused a dose-dependent reduction in acute tubular injury, long-term postinjury fibrosis, as well as improved functional recovery after ischemia-reperfusion AKI (IR-AKI). This was associated with a dose-dependent reduction in the oxidative stress marker isofuran-to-F2-isoprostane ratio, indicating that PM reduces renal oxidative damage post-AKI. PM also reduced postinjury fibrosis when administered 24 h after the initiating injury, but this was not associated with improvement in functional recovery after IR-AKI. This is the first report showing that treatment with PM reduces short- and long-term injury, fibrosis, and renal functional recovery after IR-AKI. These preclinical findings suggest that PM, which has a favorable clinical safety profile, holds therapeutic promise for AKI and, most importantly, for prevention of adverse long-term outcomes after AKI.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Piridoxamina/uso terapêutico , Complexo Vitamínico B/uso terapêutico , Injúria Renal Aguda/patologia , Animais , Relação Dose-Resposta a Droga , Fibrose , Isoprostanos/metabolismo , Túbulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estresse Oxidativo/efeitos dos fármacos , Piridoxamina/sangue , Recuperação de Função Fisiológica , Complexo Vitamínico B/sangueRESUMO
No therapies have been shown to accelerate recovery or prevent fibrosis after acute kidney injury (AKI). In part, this is because most therapeutic candidates have to be given at the time of injury and the diagnosis of AKI is usually made too late for drugs to be efficacious. Strategies to enhance post-AKI repair represent an attractive approach to address this. Using a phenotypic screen in zebrafish, we identified 4-(phenylthio)butanoic acid (PTBA), which promotes proliferation of embryonic kidney progenitor cells (EKPCs), and the PTBA methyl ester UPHD25, which also increases postinjury repair in ischemia-reperfusion and aristolochic acid-induced AKI in mice. In these studies, a new panel of PTBA analogs was evaluated. Initial screening was performed in zebrafish EKPC assays followed by survival assays in a gentamicin-induced AKI larvae zebrafish model. Using this approach, we identified UPHD186, which in contrast to UPHD25, accelerates recovery and reduces fibrosis when administered several days after ischemia-reperfusion AKI and reduces fibrosis after unilateral ureteric obstruction in mice. UPHD25 and 186 are efficiently metabolized to the active analog PTBA in liver and kidney microsome assays, indicating both compounds may act as PTBA prodrugs in vivo. UPHD186 persists longer in the circulation than UPHD25, suggesting that sustained levels of UPHD186 may increase efficacy by acting as a reservoir for renal metabolism to PTBA. These findings validate use of zebrafish EKPC and AKI assays as a drug discovery strategy for molecules that reduce fibrosis in multiple AKI models and can be administered days after initiation of injury.
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Injúria Renal Aguda/tratamento farmacológico , Butiratos/uso terapêutico , Rim/efeitos dos fármacos , Sulfetos/uso terapêutico , Injúria Renal Aguda/patologia , Animais , Butiratos/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Fibrose/tratamento farmacológico , Fibrose/patologia , Rim/patologia , Masculino , Camundongos , Sulfetos/farmacologia , Peixe-ZebraRESUMO
Despite extensive research, no therapeutic interventions have been shown to prevent AKI, accelerate recovery of AKI, or reduce progression of AKI to CKD in patients. This failure in translation has led investigators to speculate that the animal models being used do not predict therapeutic responses in humans. Although this issue continues to be debated, an important concern that has not been addressed is whether improvements in preclinical study design can be identified that might also increase the likelihood of translating basic AKI research into clinical practice using the current models. In this review, we have taken an evidence-based approach to identify common weaknesses in study design and reporting in preclinical AKI research that may contribute to the poor translatability of the findings. We focused on use of N-acetylcysteine or sodium bicarbonate for the prevention of contrast-induced AKI and use of erythropoietin for the prevention of AKI, two therapeutic approaches that have been extensively studied in clinical trials. On the basis of our findings, we identified five areas for improvement in preclinical study design and reporting. These suggested and preliminary guidelines may help improve the quality of preclinical research for AKI drug development.
Assuntos
Injúria Renal Aguda/prevenção & controle , Projetos de Pesquisa/normas , Pesquisa Translacional Biomédica/normas , Acetilcisteína/uso terapêutico , Injúria Renal Aguda/induzido quimicamente , Animais , Meios de Contraste/efeitos adversos , Modelos Animais de Doenças , Eritropoetina/uso terapêutico , Sequestradores de Radicais Livres/uso terapêutico , Humanos , Bicarbonato de Sódio/uso terapêuticoRESUMO
Phenylthiobutanoic acids (PTBAs) are a new class of histone deacetylase (HDAC) inhibitors that accelerate recovery and reduce postinjury fibrosis after ischemia-reperfusion-induced acute kidney injury. However, unlike the more common scenario in which patients present with protracted and less clearly defined onset of renal injury, this model of acute kidney injury gives rise to a clearly defined injury that begins to resolve over a short period of time. In these studies, we show for the first time that treatment with the PTBA analog methyl-4-(phenylthio)butanoate (M4PTB) accelerates recovery and reduces postinjury fibrosis in a progressive model of acute kidney injury and renal fibrosis that occurs after aristolochic acid injection in mice. These effects are apparent when M4PTB treatment is delayed 4 days after the initiating injury and are associated with increased proliferation and decreased G2/M arrest of regenerating renal tubular epithelial cells. In addition, there is reduced peritubular macrophage infiltration and decreased expression of the macrophage chemokines CX3Cl1 and CCL2. Since macrophage infiltration plays a role in promoting kidney injury, and since renal tubular epithelial cells show defective repair and a marked increase in maladaptive G2/M arrest after aristolochic acid injury, these findings suggest M4PTB may be particularly beneficial in reducing injury and enhancing intrinsic cellular repair even when administered days after aristolochic acid ingestion.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Butiratos/farmacologia , Sulfetos/farmacologia , Injúria Renal Aguda/induzido quimicamente , Animais , Ácidos Aristolóquicos/farmacologia , Butiratos/análise , Modelos Animais de Doenças , Fibrose/tratamento farmacológico , Fibrose/prevenção & controle , Inibidores de Histona Desacetilases/farmacologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Camundongos , Camundongos Biozzi , Traumatismo por Reperfusão/induzido quimicamente , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Sulfetos/análiseRESUMO
Recent studies indicate that nephron progenitor cells of the embryonic kidney are arranged in a series of compartments of an increasing state of differentiation. The earliest progenitor compartment, distinguished by expression of CITED1, possesses greater capacity for renewal and differentiation than later compartments. Signaling events governing progression of nephron progenitor cells through stages of increasing differentiation are poorly understood, and their elucidation will provide key insights into normal and dysregulated nephrogenesis, as well as into regenerative processes that follow kidney injury. In this study, we found that the mouse CITED1(+) progenitor compartment is maintained in response to receptor tyrosine kinase (RTK) ligands that activate both FGF and EGF receptors. This RTK signaling function is dependent on RAS and PI3K signaling but not ERK. In vivo, RAS inactivation by expression of sprouty 1 (Spry1) in CITED1(+) nephron progenitors results in loss of characteristic molecular marker expression and in increased death of progenitor cells. Lineage tracing shows that surviving Spry1-expressing progenitor cells are impaired in their subsequent epithelial differentiation, infrequently contributing to epithelial structures. These findings demonstrate that the survival and developmental potential of cells in the earliest embryonic nephron progenitor cell compartment are dependent on FGF/EGF signaling through RAS.