RESUMO
Exosomes are released by most cells and can be isolated from all biofluids including urine. Exosomes are small vesicles formed as part of the endosomal pathway that contain cellular material surrounded by a lipid bilayer that can be traced to the plasma membrane. Exosomes are potentially a more targeted source of material for biomarker discovery than unfractionated urine, and provide diagnostic and pathophysiological information without an invasive tissue biopsy. Cytoplasmic contents including protein, mRNA, miRNA, and lipids have all been studied within the exosomal fraction. Many prospective urinary exosomal biomarkers have been successfully identified for a variety of kidney or genitourinary tract conditions; detection of systemic conditions may also be possible. Isolation and analysis of exosomes can be achieved by several approaches, although many require specialized equipment or involve lengthy protocols. The need for timely analysis in the clinical setting has driven considerable innovation with several promising options recently emerging. Consensus on exosome isolation, characterization, and normalization procedures would resolve critical clinical translational bottlenecks for existing candidate exosomal biomarkers and provide a template for additional discovery studies.
Assuntos
Biomarcadores/urina , Exossomos , HumanosRESUMO
OBJECTIVE: The aim of this study was to examine the presence of microRNAs (miRNAs) within exosomes isolated from human saliva and to optimize and test methods for successful downstream applications. DESIGN: Exosomes isolated from fresh and frozen glandular and whole human saliva were used as a source of miRNAs. The presence of miRNAs was validated with TaqMan quantitative PCR and miRNA microarrays. RESULTS: We successfully isolated exosomes from human saliva from healthy controls and a patient with Sjögren's syndrome. microRNAs extracted from the exosomal fraction were sufficient for quantitative PCR and microarray profiling. CONCLUSIONS: The isolation of miRNAs from easily and non-invasively obtained salivary exosomes with subsequent characterization of the miRNA expression patterns is promising for the development of future biomarkers of the diagnosis and prognosis of various salivary gland pathologies.
Assuntos
Biomarcadores , Exossomos/química , MicroRNAs/metabolismo , Saliva/química , Síndrome de Sjogren/genética , Biomarcadores/análise , Estudos de Casos e Controles , Humanos , MicroRNAs/isolamento & purificação , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Sepsis remains a serious problem in critically ill patients with the mortality increasing to over half when there is attendant acute kidney injury. alpha-Melanocyte-stimulating hormone is a potent anti-inflammatory cytokine that inhibits many forms of inflammation including that with acute kidney injury. We tested whether a new alpha-melanocyte-stimulating hormone analogue (AP214), which has increased binding affinity to melanocortin receptors, improves sepsis-induced kidney injury and mortality using a cecal ligation and puncture mouse model. In the lethal cecal ligation-puncture model of sepsis, severe hypotension and bradycardia resulted and AP214 attenuated acute kidney injury of the lethal model with a bell-shaped dose-response curve. An optimum AP214 dose reduced acute kidney injury even when it was administered 6 h after surgery and it significantly improved blood pressure and heart rate. AP214 reduced serum TNF-alpha and IL-10 levels with a bell-shaped dose-response curve. Additionally; NF-kappaB activation in the kidney and spleen, and splenocyte apoptosis were decreased by the treatment. AP214 significantly improved survival in both lethal and sublethal models. We have shown that AP214 improves hemodynamic failure, acute kidney injury, mortality and splenocyte apoptosis attenuating pro- and anti-inflammatory actions due to sepsis.
Assuntos
Nefropatias/tratamento farmacológico , Sepse/complicações , alfa-MSH/análogos & derivados , Animais , Modelos Animais de Doenças , Hemodinâmica/efeitos dos fármacos , Hipotensão/tratamento farmacológico , Hipotensão/etiologia , Hipotensão/metabolismo , Interleucina-10/sangue , Rim/efeitos dos fármacos , Rim/metabolismo , Nefropatias/etiologia , Nefropatias/metabolismo , Fígado/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos , NF-kappa B/metabolismo , Neutropenia/tratamento farmacológico , Neutropenia/etiologia , Neutropenia/metabolismo , Baço/efeitos dos fármacos , Baço/metabolismo , Fator de Necrose Tumoral alfa/sangue , alfa-MSH/farmacologia , alfa-MSH/uso terapêuticoRESUMO
Observational studies suggest improvements with frequent hemodialysis (HD), but its true efficacy and safety remain uncertain. The Frequent Hemodialysis Network Trials Group is conducting two multicenter randomized trials of 250 subjects each, comparing conventional three times weekly HD with (1) in-center daily HD and (2) home nocturnal HD. Daily HD will be delivered for 1.5-2.75 h, 6 days/week, with target eK(t)/V(n) > or = 0.9/session, whereas nocturnal HD will be delivered for > or = 6 h, 6 nights/week, with target stdK(t)/V of > or = 4.0/week. Subjects will be followed for 1 year. The composite of mortality with the 12-month change in (i) left ventricular mass index (LVMI) by magnetic resonance imaging, and (ii) SF-36 RAND Physical Health Composite (PHC) are specified as co-primary outcomes. The seven main secondary outcomes are between group comparisons of: change in LVMI, change in PHC, change in Beck Depression Inventory score, change in Trail Making Test B score, change in pre-HD serum albumin, change in pre-HD serum phosphorus, and rates of non-access hospitalization or death. Changes in blood pressure and erythropoiesis will also be assessed. Safety outcomes will focus on vascular access complications and burden of treatment. Data will be obtained on the cost of delivering frequent HD compared to conventional HD. Efforts will be made to reduce bias, including blinding assessment of subjective outcomes. Because no large-scale randomized trials of frequent HD have been previously conducted, the first year has been designated a Vanguard Phase, during which feasibility of randomization, ability to deliver the interventions, and adherence will be evaluated.
Assuntos
Hipertrofia Ventricular Esquerda/prevenção & controle , Qualidade de Vida , Diálise Renal/métodos , Protocolos Clínicos , Interpretação Estatística de Dados , Humanos , Diálise Renal/efeitos adversos , Diálise Renal/economia , Projetos de Pesquisa , Fatores de Tempo , Resultado do Tratamento , Recusa do Paciente ao TratamentoRESUMO
Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We aimed to discover biomarkers in urinary exosomes to detect acute kidney injury (AKI), which has a high mortality and morbidity. Animals were injected with cisplatin. Urinary exosomes were isolated by differential centrifugation. Protein changes were evaluated by two-dimensional difference in gel electrophoresis and changed proteins were identified by mass spectrometry. The identified candidate biomarkers were validated by Western blotting in individual urine samples from rats subjected to cisplatin injection; bilateral ischemia and reperfusion (I/R); volume depletion; and intensive care unit (ICU) patients with and without AKI. We identified 18 proteins that were increased and nine proteins that were decreased 8 h after cisplatin injection. Most of the candidates could not be validated by Western blotting. However, exosomal Fetuin-A increased 52.5-fold at day 2 (1 day before serum creatinine increase and tubule damage) and remained elevated 51.5-fold at day 5 (peak renal injury) after cisplatin injection. By immunoelectron microscopy and elution studies, Fetuin-A was located inside urinary exosomes. Urinary Fetuin-A was increased 31.6-fold in the early phase (2-8 h) of I/R, but not in prerenal azotemia. Urinary exosomal Fetuin-A also increased in three ICU patients with AKI compared to the patients without AKI. We conclude that (1) proteomic analysis of urinary exosomes can provide biomarker candidates for the diagnosis of AKI and (2) urinary Fetuin-A might be a predictive biomarker of structural renal injury.
Assuntos
Injúria Renal Aguda/urina , Proteínas Sanguíneas/urina , Proteômica/métodos , Traumatismo por Reperfusão/urina , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Biomarcadores/urina , Membrana Celular/metabolismo , Cisplatino/efeitos adversos , Cisplatino/farmacologia , Feminino , Humanos , Rim/efeitos dos fármacos , Rim/lesões , Rim/patologia , Masculino , Pessoa de Meia-Idade , Modelos Animais , Ratos , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/patologia , alfa-2-Glicoproteína-HS , alfa-Fetoproteínas/urinaRESUMO
Sepsis is one of the common causes of acute renal failure (ARF). The objective of this study was to identify new biomarkers and therapeutic targets. We present a new rat model of sepsis-induced ARF based on cecal ligation and puncture (CLP). We used this model to find urinary proteins which may be potential biomarkers and/or drug targets. Aged rats were treated with fluids and antibiotics after CLP. Urinary proteins from septic rats without ARF and urinary proteins from septic rats with ARF were compared by difference in-gel electrophoresis (DIGE). CLP surgery elevated interleukin (IL)-6 and IL-10 serum cytokines and blood nitrite compared with sham-operated rats. However, there was a range of serum creatinine values at 24 h (0.4-2.3 mg/dl) and only 24% developed ARF. Histology confirmed renal injury in these rats. Forty-nine percent of rats did not develop ARF. Rats without ARF also had less liver injury. The mortality rate at 24 h was 27% but was increased by housing the post-surgery rats in metabolic cages. Creatinine clearance and urine output 2-8 h after CLP was significantly reduced in rats which died within 24 h. Using DIGE we identified changes in a number of urinary proteins including albumin, brush-border enzymes (e.g., meprin-1-alpha) and serine protease inhibitors. The meprin-1-alpha inhibitor actinonin prevented ARF in aged mice. In summary, we describe a new rat model of sepsis-induced ARF which has a heterogeneous response similar to humans. This model allowed us to use DIGE to find changes in urinary proteins and this approach identified a potential biomarker and drug target - meprin-1-alpha.
Assuntos
Injúria Renal Aguda/urina , Modelos Animais de Doenças , Proteômica/métodos , Ratos Sprague-Dawley , Sepse/urina , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/patologia , Animais , Antibacterianos/farmacologia , Biomarcadores/urina , Ceco , Citocinas/sangue , Desenho de Fármacos , Eletroforese/métodos , Ácidos Hidroxâmicos/farmacologia , Rim/patologia , Ligadura , Fígado/patologia , Masculino , Metaloendopeptidases/urina , Camundongos , Camundongos Endogâmicos C57BL , Nitritos/sangue , Ratos , Sepse/complicações , Sepse/tratamento farmacológicoRESUMO
Acute kidney injury (AKI) occurs in about half of patients in septic shock and the mortality of AKI with sepsis is extremely high. An effective therapeutic intervention is urgently required. Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors that also have pleiotropic actions. They have been reported to increase the survival of septic or infectious patients. But the effect of simvastatin, a widely used statin, on sepsis-induced AKI is unknown. The effects of simvastatin and tumor necrosis factor (TNF)-alpha neutralizing antibody were studied in a clinically relevant model of sepsis-induced AKI using cecal ligation and puncture (CLP) in elderly mice. Simvastatin significantly improved CLP-induced mortality and AKI. Simvastatin attenuated CLP-induced tubular damage and reversed CLP-induced reduction of intrarenal microvascular perfusion and renal tubular hypoxia at 24 h. Simvastatin also restored towards normal CLP-induced renal vascular protein leak and serum TNF-alpha. Neither delayed simvastatin therapy nor TNF-alpha neutralizing antibody improved CLP-induced AKI. Simvastatin improved sepsis-induced AKI by direct effects on the renal vasculature, reversal of tubular hypoxia, and had a systemic anti-inflammatory effect.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Circulação Renal/efeitos dos fármacos , Sepse/tratamento farmacológico , Sinvastatina/administração & dosagem , Injúria Renal Aguda/etiologia , Animais , Anticorpos/farmacologia , Vasos Sanguíneos/efeitos dos fármacos , Preparações de Ação Retardada/administração & dosagem , Hipóxia/tratamento farmacológico , Túbulos Renais/irrigação sanguínea , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , Lipídeos/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microcirculação/efeitos dos fármacos , Permeabilidade , Sepse/complicações , Sepse/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Toll-like receptors (TLRs) are important in sepsis. Myeloid differentiation factor 88 (MyD88) is a key molecule involved in signal transduction by multiple TLRs. The objective of this study was to investigate the contribution of TLR4 and MyD88 to acute renal failure (ARF) induced by polymicrobial sepsis. Liver dysfunction and apoptosis in the spleen contribute to sepsis severity after cecal ligation and puncture (CLP). Therefore, we also investigated liver injury and splenic apoptosis. We used a mouse model of sepsis-induced ARF using CLP to generate polymicrobial sepsis. Despite fluid and antibiotic resuscitation the mice developed multi-organ failure, including ARF, which resembles human sepsis. We investigated the role of the TLR4 receptor by comparing C3H/HeJ mice (which lack TLR4) with C3H/He0UJ normal controls. The role of MyD88 was investigated by comparing MyD88 knockout mice (MyD88(-/-)) with wild-type controls. Following CLP, mice lacking TLR4 and wild-type mice both developed comparable ARF. However, MyD88(-/-) mice did not develop ARF compared to wild-type controls. In contrast, MyD88(-/-) mice developed liver injury comparable to wild type. After CLP, MyD88(-/-) mice had significantly reduced apoptosis in the spleen compared with wild type. Apoptosis was not detected in the kidney of wild-type or MyD88(-/-) mice after CLP. In summary, ARF induced by polymicrobial sepsis is dependent on MyD88, but not TLR4. The absence of MyD88 dissociates ARF from liver injury; liver injury is MyD88-independent. There was MyD88-dependent apoptosis in the spleen, but no apoptosis in the kidney. MyD88 may be a good drug target for some, but not all, organ dysfunctions following sepsis.
Assuntos
Injúria Renal Aguda/etiologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Falência Hepática Aguda/etiologia , Insuficiência de Múltiplos Órgãos/etiologia , Sepse/complicações , Injúria Renal Aguda/patologia , Injúria Renal Aguda/fisiopatologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose , Feminino , Falência Hepática Aguda/patologia , Falência Hepática Aguda/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide , Baço/patologiaRESUMO
Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80 degrees C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20 degrees C caused a major loss in exosomes compared to fresh urine. In contrast, recovery after freezing at -80 degrees C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80 degrees C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80 degrees C with extensive vortexing after thawing maximizes the recovery of urinary exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts.
Assuntos
Biomarcadores/urina , Criopreservação , Proteínas de Membrana/urina , Fragmentos de Peptídeos/urina , Simportadores/urina , Western Blotting , Criopreservação/instrumentação , Criopreservação/métodos , Citosol/química , Eletroforese em Gel de Poliacrilamida , Humanos , Proteínas de Membrana/isolamento & purificação , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/isolamento & purificação , Inibidores de Proteases/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Simportadores de Cloreto de Sódio-Potássio/genética , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Acute renal failure (ARF) is caused by ischemic and nephrotoxic insults acting alone or in combination. Anti-inflammatory agents have been shown to decrease renal ischemia-reperfusion and cisplatin-induced injury and leukocyte infiltration. Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine that inhibits inflammatory and cytotoxic pathways implicated in acute renal injury. Therefore, we sought to determine if IL-10 inhibits acute renal injury. METHODS: The effects of IL-10 were studied in mice following cisplatin administration and bilateral renal ischemia-reperfusion, in a rat model of renal transplantation, and in cultured mouse cortical tubule cells. RESULTS: IL-10 significantly decreased renal injury following cisplatin administration and following renal ischemia/reperfusion. Delay of IL-10 treatment for one hour after cisplatin also significantly inhibited renal damage. IL-10 and alpha-melanocyte stimulating hormone (alpha-MSH) increased recovery following transplantation of a kidney subjected to warm ischemia. To explore the mechanism of action of IL-10, its effects were measured on mediators of leukocyte trafficking and inducible nitric oxide synthase (NOS-II). IL-10 inhibited cisplatin and ischemia-induced increases in mRNA for tumor necrosis factor-alpha (TNF-alpha), intercellular adhesion molecule-1 (ICAM-1), and NOS-II. IL-10 also inhibited staining for markers of apoptosis and cell cycle activity following cisplatin administration, and nitric oxide production in cultured mouse cortical tubules. CONCLUSIONS: IL-10 protects against renal ischemic and cisplatin-induced injury. IL-10 may act, in part, by inhibiting the maladaptive activation of genes that cause leukocyte activation and adhesion, and induction of iNOS.
Assuntos
Cisplatino/farmacologia , Interleucina-10/farmacologia , Isquemia/patologia , Rim/efeitos dos fármacos , Rim/patologia , Circulação Renal , Animais , Células Cultivadas , Interleucina-10/administração & dosagem , Rim/fisiopatologia , Transplante de Rim , Túbulos Renais/citologia , Túbulos Renais/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Preservação Biológica , Ratos , Ratos Endogâmicos Lew , Traumatismo por Reperfusão/patologia , Fatores de Tempo , alfa-MSH/farmacologiaRESUMO
The intrarenal localization and role of the V1a vasopressin receptor in body fluid homeostasis are unclear. We investigated the intranephron localization of V1a receptor mRNA and protein using reverse transcription (RT)-competitive polymerase chain reaction (PCR) and immunohistochemistry with a specific polyclonal antibody. To determine whether the V1a receptor is involved in the regulation of acid-base balance, we also examined the effects of acute and chronic metabolic acidosis and dehydration on V1a receptor expression. V1a mRNA was expressed most abundantly in the cortical collecting ducts (CCD) and decreased in the deeper CD. Expression in the glomeruli and thick ascending limbs was low. The immunohistochemical study revealed the presence of the V1a receptor in the glomeruli, the thick ascending limbs and the CD. Dehydration decreased V1a mRNA expression in the CD. Chronic metabolic acidosis increased V1a receptor mRNA expression in the CD but decreased V2 receptor mRNA expression. Western blot analysis revealed up-regulation of the V1a receptor protein in chronic metabolic acidosis. Incubation of microdissected CCD or outer medullary CD (OMCD) in a low-pH (or or low-HCO3-) medium increased the levels of V1a receptor mRNA but decreased V2 receptor mRNA expression. Incubating OMCD with arginine vasopressin (AVP) and the V1a receptor antagonist (OPC21268) increased V2 receptor mRNA expression compared with incubation with AVP alone. These data suggest that V1a receptors are present primarily in the principal and intercalated cells in the CD and that these receptors are involved in the regulation of water and acid-base balance.
Assuntos
Acidose/metabolismo , Desidratação/metabolismo , Néfrons/metabolismo , Receptores de Vasopressinas/metabolismo , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos , Arginina Vasopressina/análogos & derivados , Arginina Vasopressina/farmacologia , Bicarbonatos/farmacologia , Regulação para Baixo , Homeostase , Imuno-Histoquímica , Masculino , Néfrons/anatomia & histologia , Piperidinas/farmacologia , Reação em Cadeia da Polimerase/métodos , Quinolonas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Vasopressinas/genética , Regulação para Cima , ATPases Vacuolares Próton-Translocadoras/metabolismoAssuntos
National Institutes of Health (U.S.) , Análise de Sequência com Séries de Oligonucleotídeos/normas , Animais , Bases de Dados Factuais , Genoma Humano , Humanos , Maryland , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos/economia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Estados UnidosRESUMO
BACKGROUND: The next phase of the molecular revolution will bring functional genomics down to the level of individual cells in a tissue. Laser capture microdissection (LCM) coupled with reverse transcription-polymerase chain reaction (RT-PCR) can measure gene expression in normal, cancerous, injured, or fibrotic tissue. Nevertheless, targeting of specific cells may be difficult using routine morphologic stains. Immunohistochemistry can identify cells with specific antigens; however, exposure to aqueous solutions destroys 99% of the mRNA. Consequently, there is an overwhelming need to identify specific tissue cells for LCM without mRNA loss. We report on a rapid immunofluorescent LCM (IF-LCM) procedure that allows targeted analysis of gene expression. METHODS: A LCM microscope was outfitted for epifluorescence and light level video microscopy. Heat filters were added to shield the image intensifier from the laser. Frozen sections were fluorescently labeled by a rapid one minute incubation with anti-Tamm-Horsfall antibody and an ALEXA-linked secondary antibody. Fluorescently labeled thick ascending limb (TAL) cells were detected by low light level video microscopy, captured by LCM, and mRNA was analyzed by RT-PCR for basic amino acid transporter, Tamm-Horsfall protein, and aquaporin-2. RESULTS: The immunofluorescently identified TAL could be cleanly microdissected without contamination from surrounding tubules. The recovery of RNA following rapid immunofluorescence staining was similar to that obtained following hematoxylin and eosin staining, as assessed by RT-PCR for malate dehydrogenase. CONCLUSIONS: We conclude that the new apparatus and method for the immunofluorescent labeling of tissue cells targeted for LCM can isolate pure populations of targeted cells from a sea of surrounding cells with highly acceptable preservation of mRNA. Since the TAL is minimally injured following ischemia, identification of the different responses between TAL and surrounding tissue in damaged kidneys may provide new therapeutic targets or agents for the treatment of acute renal failure.
Assuntos
Dissecação/métodos , Lasers , Alça do Néfron/citologia , Microscopia de Vídeo/métodos , RNA Mensageiro/análise , Laranja de Acridina , Animais , Corantes , Dissecação/instrumentação , Amarelo de Eosina-(YS) , Feminino , Imunofluorescência , Expressão Gênica , Hematoxilina , Citometria por Imagem/instrumentação , Citometria por Imagem/métodos , Alça do Néfron/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Vídeo/instrumentação , Sondas de Oligonucleotídeos , Óptica e Fotônica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
UNLABELLED: Analysis of segmental renal gene expression by laser capture microdissection. BACKGROUND: The study of normal renal physiology has been greatly aided by microdissection techniques that have delineated the exceptional functional and cellular heterogeneity both along the nephron and between different nephron populations. These techniques are not widely used to study renal injury as microdissection is difficult because of tissue necrosis or fibrosis. We developed a procedure to detect specific gene expression in specific locations of the kidney in histologic sections. METHODS: The anatomic specificity of laser capture microdissection (LCM) was employed with the sensitivity of reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: LCM/RT-PCR detected mRNA for podoplanin in 2% of a single glomerulus, rat basic amino acid transporter in 6% of a single cross-section of proximal straight tubule, and renin in eight proximal convoluted tubule cross-sections. LCM/RT-PCR could isolate pure populations of proximal convoluted tubules, proximal straight tubules, and thick ascending limbs from renal histologic sections, although pure collecting ducts could not be isolated. LCM/RT-PCR localized ischemia-reperfusion-induced induction of KC/interleukin-8 primarily to the medullary thick ascending limb, and detected transforming growth factor-beta (TGF-beta) mRNA in glomeruli of a patient with membranous glomerulonephropathy. CONCLUSIONS: When used with an appropriate laser spot size, LCM/RT-PCR can measure gene expression in glomeruli or specific parts of the nephron and can study alterations in steady-state mRNA levels in animal models of renal disease. The applications, limitations, and refinements of this approach are discussed.
Assuntos
Expressão Gênica , Glomérulos Renais/metabolismo , Túbulos Renais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Sequência de Bases , Primers do DNA , Feminino , Humanos , Interleucina-8/genética , Lasers , Masculino , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fator de Crescimento Transformador beta/genéticaRESUMO
BACKGROUND AND PURPOSE: Potential drugs for human acute renal failure are often tested in an animal model of renal ischemia/reperfusion injury. Analgesics are often not given after surgery because of concerns that they would alter renal function. Therefore, we tested whether postoperative analgesia would alter animal health or affect the degree of renal injury. METHODS: Mice were subjected to either 32 or 37 minutes of renal ischemia, given two or six doses of buprenorphine or vehicle at 12-hour intervals, and followed for 72 hours. In some animals, we measured body temperature and physical activity by use of telemetry. RESULTS: Animals treated with buprenorphine recovered more rapidly from surgery based on postoperative activity, and had a small but not significant tendency for faster restoration of normal body temperature. Animals treated with buprenorphine had less weight loss after 37 minutes of ischemia. Buprenorphine given after surgery did not influence the degree of renal injury after ischemia/reperfusion. CONCLUSIONS: Buprenorphine should be given after renal ischemia-reperfusion surgery because administration of the proper analgesic improved animal health without interfering with the renal ischemia/reperfusion model. Analgesic treatment at the time of the operation and 12 hours after was sufficient. Buprenorphine may reduce the post-surgical stress response, and thus potentially improve the specificity of testing for drugs that reduce or treat renal injury.
Assuntos
Analgésicos Opioides/farmacologia , Buprenorfina/farmacologia , Rim/irrigação sanguínea , Traumatismo por Reperfusão/fisiopatologia , Procedimentos Cirúrgicos Operatórios , Animais , Temperatura Corporal/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Atividade Motora/efeitos dos fármacosRESUMO
BACKGROUND: The volume of urea distribution (V) is usually derived from single-pool variable volume urea kinetics. A theoretical analysis has shown that modeled single-pool V (Vsp) is overestimated when the urea reduction ratio (URR) is greater than 65 to 70% and is underestimated when the URR is less than 65%. The "true" volume derived from double-pool kinetics (Vdp) does not exhibit this effect. An equation has been derived to adjust Vsp to the expected Vdp. METHODS: To validate these theoretical predictions, we examined data from the Hemodialysis (HEMO) Study to assess the performance of Vdp as estimated from Vsp using the previously published prediction equation. For increased precision, both Vsp and Vdp were factored by anthropometric volume (Va). Patients were first dialyzed with a target equilibrated dialysis dose (eKt/V) of 1.45 during a baseline period and were then randomly assigned to eKt/V targets of either 1. 05 (a URR of approximately 67%) or 1.45 (a URR of approximately 75%). A blood sample was obtained one hour after starting dialysis during one dialysis in each patient. RESULTS: Vsp/Va was (mean +/- SD) 1.014 +/- 0.127 in 795 patients during the baseline period when the URR was approximately 1.45. During the first modeled dialysis after randomization, the Vsp/Va fell to 0.961 +/- 0.138 in the group with an eKt/V target of 1.05, but did not change significantly under the high eKt/V goal. The correction of Vsp to Vdp using the prediction equation resulted in a Vdp/Va ratio of 0.96 to 0.98 in all three circumstances without significant differences. When a blood sample was drawn one hour after starting dialysis, the apparent Vsp/Va ratio at one hour was much lower at 0.708 +/- 0.139. However, the mean Vdp/Va ratio, computed using the correction equation, was 0.968 +/- 0.322, which was similar to the Vdp/Va ratio calculated from the postdialysis blood urea nitrogen. CONCLUSIONS: These data suggest that the previously derived formula for adjusted Vsp is valid experimentally. The Vsp/Vdp correction should be useful for prescribing hemodialysis with either a very low Kt/V (for example, daily and early incremental dialysis) or a very high Kt/V.
Assuntos
Diálise Renal , Ureia/metabolismo , Humanos , Modelos BiológicosRESUMO
BACKGROUND: The postdialysis blood urea nitrogen (BUN; Ct) is a pivotal parameter for assessing hemodialysis adequacy by conventional blood-side methods, but Ct is relatively unstable because of hemodialysis-induced disequilibrium. The uncertainty associated with this method is potentially reduced or eliminated by measuring urea removed on the dialysate side, a more direct approach that can determine adequacy from the fraction of urea removed and by substituting an estimate of the equilibrated postdialysis BUN (Ceq) for Ct. For a patient with a known urea volume (V), Ceq, the equilibrated Kt/V (eKt/V), and the solute removal index (SRI) can be calculated from the predialysis BUN (C0), total urea nitrogen removed (A), and V from simple mass balance calculations (dialysate/volume method). However, a theoretical error analysis showed that relatively small errors in A, C0, or V are magnified when SRI or eKt/V is calculated using this method, especially at higher eKt/V values (for example, if eKt/V = 1.4 per dialysis, a 7% dialysate collection error causes a 20% error in eKt/V). METHODS: During three to four baseline dialyses in each of 39 patients enrolled in the pilot phase of the HEMO Study, "A" was measured using an instrument that sampled dialysate frequently (Biostat), and V was calculated from A, C0, and Ceq (median CV for V = 5.6%). The mean V was then applied to the dialysate/volume method to estimate eKt/V and SRI during two to five subsequent dialyses per patient (comparison dialyses). The accuracy and precision of these estimates were assessed by comparing them with eKt/V and SRI derived from a direct measurement of Ceq drawn 30 minutes after dialysis (reference method), from mathematical curve-fitting of sequential dialysate urea concentrations (dialysate curve-fit method), and from another blood-side method that estimates eKt/V from single pool Kt/V and the fractional rate of solute removal (rate method): eKt/V = spKt/V - 0.6.K/V + 0.03. RESULTS: During 128 comparison dialyses, median absolute errors for calculated eKt/V compared with the reference method were 0.169, 0.061, and 0.071 for the dialysate/volume method, the rate method, and the dialysate curve-fitting method, respectively. The corresponding correlation coefficients were 0.47, 0.88, and 0.81. For SRI, median absolute errors were 0.044, 0.018, and 0.027, and the correlation coefficients were 0.54, 0.85, and 0.74 for the three methods. CONCLUSIONS: The precision of eKt/V and SRI measurements was significantly lower for the dialysate/volume method compared with the blood-side methods. Inclusion of the dialysate curve analysis provided by the Biostat restored precision to the dialysate method to a level comparable to that of the blood-side methods. New techniques employing dialysate urea analysis should include a concentration profile to avoid these inherent methodological errors and assure the accuracy of eKt/V and SRI.
Assuntos
Diálise Renal/métodos , Ureia/análise , Ureia/sangue , Feminino , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Projetos PilotoRESUMO
Acute renal failure is a life threatening illness whose mortality has remained high since the introduction of hemodialysis 25 years ago, despite advances in supportive care. Acute renal failure is an extremely morbid and costly disorder with a significant proportion of patients progressing to end-stage renal disease requiring dialysis. To the nephrologist, acute renal failure remains an extremely frustrating disease, because the pathophysiology is not well understood and the limited therapeutic options force the nephrologist to sit on the sidelines and wait for renal function to return. For example, dialysis remains the only FDA-approved treatment for acute renal failure, but dialysis may also cause renal injury that prolongs renal failure. The purpose of this perspective is to understand the results of the recent, largely negative, clinical trials in view of recent advances in the epidemiology of ARF. This review will also discuss diagnostic tools, strategies for improved design of clinical trials, and other therapeutic interventions that will be needed to properly treat acute renal failure in the 21st century.
Assuntos
Injúria Renal Aguda/terapia , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/epidemiologia , Injúria Renal Aguda/fisiopatologia , Humanos , Incidência , Terapia de Substituição RenalRESUMO
BACKGROUND: The apical potassium (K+) channels mediate K+ recycling in thick ascending limb (TAL) and K+ secretion in cortical collecting duct (CCD). Recently, the cDNAs for a family of renal K+ channels, ROMK1, -2 and -3, were identified. Based on the biophysical properties and mRNA distribution, it is believed that these ROMK cDNAs encode the apical K+ channels of TAL and CCD. However, the information for cellular and subcellular localization of the ROMK proteins in these tubules is still not available. METHODS: Paraffin or frozen kidney sections from adult Sprague-Dawley rats were stained by polyclonal antibodies against the N- and C-terminal domain of ROMK. Immunoreactive staining was visualized by color development from horseradish peroxidase reaction. Membrane homogenates from kidney were analyzed by Western blot analysis. RESULTS: The polyclonal antibodies against cytoplasmic epitope of ROMK recognized a approximately 42 kD protein in the membrane homogenates from kidney, but not from liver. Staining by immunocytochemistry revealed that ROMK channels were localized to the apical membranes of the distal nephron in cortex and outer medulla, including thick ascending limb and collecting tubule. ROMK staining was absent in glomerulus, proximal tubule and inner medulla. Double staining of the tissue section with both ROMK-specific and H+-ATPase-specific antibodies revealed labeling of ROMK in the principal cells of the collecting tubules. CONCLUSIONS: These results further strengthen the idea that ROMK channels play important roles in the recycling of K+ in TAL and the secretion of K+ in CCD.