RESUMEN
Acute kidney injury (AKI) initiated by sepsis remains a thorny problem despite recent advancements in its clinical management. Having been found to be activated during AKI, fibroblast growth factor-inducible molecule 14 (Fn14) may be a potential therapeutic target because of its involvement in the molecular basis of injury. Here, we report that LPS induces apoptosis of mouse cortical tubule cells mediated by Fn14, for which simultaneous Toll-like receptor (TLR)4 activation is required. Mechanistically, TLR4 activation by lipopolysaccharide, through disassociating E3 ligase SCFFbxw7α from Fn14, dismantles Lys48-linked polyubiquitination of Fn14 and stabilizes it. Pharmacological deactivation of Fn14 with monoclonal antibody ITEM-2 provides effective protection against lethal sepsis and AKI in mice. Our study underscores an adaptive mechanism whereby TLR4 regulates SCFFbxw7α-dependent Fn14 stabilization during inflammatory tubular damage and further supports investigation of targeting Fn14 in clinical trials of patients with septic AKI.
Asunto(s)
Lesión Renal Aguda/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Túbulos Renales/metabolismo , Macrófagos/metabolismo , Sepsis/complicaciones , Receptor de TWEAK/metabolismo , Lesión Renal Aguda/genética , Lesión Renal Aguda/microbiología , Lesión Renal Aguda/patología , Animales , Apoptosis , Modelos Animales de Enfermedad , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Túbulos Renales/microbiología , Túbulos Renales/patología , Macrófagos/microbiología , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Estabilidad Proteica , Células RAW 264.7 , Sepsis/microbiología , Transducción de Señal , Receptor de TWEAK/genética , Receptor Toll-Like 4/metabolismoRESUMEN
Females across their lifespan and certain male populations are susceptible to urinary tract infections (UTI). The influence of female vs. male sex on UTI is incompletely understood, in part because preclinical modeling has been performed almost exclusively in female mice. Here, we employed established and new mouse models of UTI with uropathogenic Escherichia coli (UPEC) to investigate androgen influence on UTI pathogenesis. Susceptibility to UPEC UTI in both male and female hosts was potentiated with 5α-dihydrotestosterone, while males with androgen receptor deficiency and androgenized females treated with the androgen receptor antagonist enzalutamide were protected from severe pyelonephritis. In androgenized females and in males, UPEC formed dense intratubular, biofilm-like communities, some of which were sheltered from infiltrating leukocytes by the tubular epithelium and by peritubular fibrosis. Abscesses were nucleated by small intratubular collections of UPEC first visualized at five days postinfection and briskly expanded over the subsequent 24 hours. Male mice deficient in Toll-like receptor 4, which fail to contain UPEC within abscesses, were susceptible to lethal dissemination. Thus, androgen receptor activation imparts susceptibility to severe upper-tract UTI in both female and male murine hosts. Visualization of intratubular UPEC communities illuminates early renal abscess pathogenesis and the role of abscess formation in preventing dissemination of infection. Additionally, our study suggests that androgen modulation may represent a novel therapeutic route to combat recalcitrant or recurrent UTI in a range of patient populations.
Asunto(s)
Absceso/patología , Antagonistas de Receptores Androgénicos/farmacología , Andrógenos/farmacología , Túbulos Renales/patología , Pielonefritis/patología , Receptores Androgénicos/metabolismo , Absceso/microbiología , Antagonistas de Receptores Androgénicos/uso terapéutico , Animales , Benzamidas , Dihidrotestosterona/farmacología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades/microbiología , Susceptibilidad a Enfermedades/patología , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/patología , Femenino , Humanos , Túbulos Renales/efectos de los fármacos , Túbulos Renales/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nitrilos , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/farmacología , Feniltiohidantoína/uso terapéutico , Pielonefritis/tratamiento farmacológico , Pielonefritis/microbiología , Factores Sexuales , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Resultado del Tratamiento , Infecciones Urinarias/tratamiento farmacológico , Infecciones Urinarias/microbiología , Infecciones Urinarias/patología , Escherichia coli Uropatógena/patogenicidadRESUMEN
The pathogenesis and therapy of Shigatoxin 2 (Stx2)-mediated kidney failure remain controversial. Our aim was to test whether, during an infection with Stx2-producing E. coli (STEC), Stx2 exerts direct effects on renal tubular epithelium and thereby possibly contributes to acute renal failure. Mice represent a suitable model because they, like humans, express the Stx2-receptor Gb3 in the tubular epithelium but, in contrast to humans, not in glomerular endothelia, and are thus free of glomerular thrombotic microangiopathy (TMA). In wild-type mice, Stx2 caused acute tubular dysfunction with consequent electrolyte disturbance, which was most likely the cause of death. Tubule-specific depletion of Gb3 protected the mice from acute renal failure. In vitro, Stx2 induced secretion of proinflammatory cytokines and apoptosis in human tubular epithelial cells, thus implicating a direct effect of Stx2 on the tubular epithelium. To correlate these results to human disease, kidney biopsies and outcome were analysed in patients with Stx2-associated kidney failure (n = 11, aged 22-44 years). The majority of kidney biopsies showed different stages of an ongoing TMA; however, no glomerular complement activation could be demonstrated. All biopsies, including those without TMA, showed severe acute tubular damage. Due to these findings, patients were treated with supportive therapy without complement-inhibiting antibodies (eculizumab) or immunoadsorption. Despite the severity of the initial disease [creatinine 6.34 (1.31-17.60) mg/dl, lactate dehydrogenase 1944 (753-2792) U/l, platelets 33 (19-124)/nl and haemoglobin 6.2 (5.2-7.8) g/dl; median (range)], all patients were discharged after 33 (range 19-43) days with no neurological symptoms and no dialysis requirement [creatinine 1.39 (range 0.84-2.86) mg/dl]. The creatinine decreased further to 0.90 (range 0.66-1.27) mg/dl after 24 months. Based on these data, one may surmise that acute tubular damage represents a separate pathophysiological mechanism, importantly contributing to Stx2-mediated acute kidney failure. Specifically in young adults, an excellent outcome can be achieved by supportive therapy only.
Asunto(s)
Lesión Renal Aguda/patología , Infecciones por Escherichia coli/patología , Toxina Shiga II/metabolismo , Escherichia coli Shiga-Toxigénica/patogenicidad , Lesión Renal Aguda/microbiología , Lesión Renal Aguda/terapia , Adulto , Animales , Biopsia , Línea Celular , Estudios de Cohortes , Creatinina/metabolismo , Modelos Animales de Enfermedad , Epitelio/microbiología , Epitelio/patología , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/terapia , Femenino , Globósidos/metabolismo , Humanos , Túbulos Renales/microbiología , Túbulos Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Toxina Shiga II/genética , Microangiopatías Trombóticas , Resultado del Tratamiento , Adulto JovenRESUMEN
The macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is deregulated in acute kidney injury (AKI) through an unknown mechanism. In the present study, we used a previously described mouse model of ascending urinary tract infection in which uropathogenic Escherichia coli (UPEC) were transurethrally inoculated to induce kidney infections. Here, we show that urinary MIF was upregulated during AKI while MIF was abundantly expressed in the renal cortical tubules and that UPEC infection caused a decrease in tubular MIF. Infections with UPEC in vitro caused MIF release in a cell type-dependent manner, which was independent of receptor-mediated internalization, signal transduction, and transcription. Indeed, UPEC infection-induced necrotic cell death in vitro and in vivo correlated with extracellular acidification and processed MIF secretion. These data suggest that MIF is released by necrotic renal cortical tubular cells during UPEC infection.
Asunto(s)
Infecciones por Escherichia coli/patología , Corteza Renal/patología , Túbulos Renales/patología , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Infecciones Urinarias/patología , Escherichia coli Uropatógena/fisiología , Ácidos/metabolismo , Lesión Renal Aguda/microbiología , Lesión Renal Aguda/patología , Lesión Renal Aguda/orina , Animales , Muerte Celular , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/orina , Femenino , Humanos , Corteza Renal/microbiología , Corteza Renal/ultraestructura , Túbulos Renales/microbiología , Túbulos Renales/ultraestructura , Factores Inhibidores de la Migración de Macrófagos/orina , Ratones , Ratones Endogámicos C57BL , Necrosis , Especificidad de Órganos , Transducción de Señal , Transcripción Genética , Infecciones Urinarias/microbiología , Infecciones Urinarias/orinaRESUMEN
OBJECTIVE: To study the damage of nanobacteria on HK-2 cells, the possible principles, the effect of crystals (COM) adhering to HK-2 cells after the damage. METHODS: Four groups were chosen for the study: control group, NB group, nHAP group and COM group. Morphological changes of the HK-2 cells were observed after HE stain and with TEM after 12 hours and 24 hours. Meanwhile, the levels of H2O2, LDH, MDA and ATPases were surveyed after 6 hours,12 hours and 24 hours, respectively. And 6, 12, and 24 hours later, COM crystals were mixed into the culture fluids of each group. Then phalloidin-FITC was used to finish fluorescent staining of the cells. At last, the adhering effects of each group with the laser scanning confocal fluorescence microscope were observed and contrasted. RESULTS: After HE stain and with TEM: in NB and nHAP group, the shape of the cells changed, brush borders were arranged in disorder, vacuoles formed in the kytoplasms, the mitochondria became swelled up, the karyotheca dissolved and the nucleolus disappeared in some cells. After 24 hours, in NB group, the number of the cells in which the karyotheca dissolved was more than that in nHAP group. After 12 and 24 hours, the level of H2O2 in NB group was higher than that in control group and nHAP group; After 6 and 24 hours, the level of MDA in NB group was higher than that in control group and nHAP group; At each time point, there was no significant difference in the level of LDH between control group, nHAP group and NB group; After 12 hours, the activities of Na+/K+ ATPases in NB group and nHAP group were lower than those in control group. And after 24 hours, the activity of Na+/K+ ATPases in NB group was lower than that in control group; After 12 and 24 hours, the activities of Ca2+/Mg2+ ATPases in NB group was lower than those in control group. After 12 hours, the activity of Ca2+/Mg2+ ATPases in nHAP group was lower than that in control group. The observation with the laser scanning confocal fluorescence microscope: after 12 hours, showed that the number of the crystals adhering to the cells in NB group and COM group increased, and in COM group, some crystals had entered the cells; after 24 hours, the adhering effects of the crystals in NB and COM group were similar to those after 12 hours, but the number of adhered crystals was more than that after 12 hours; At each time point, there was no significant change in control and nHAP groups. CONCLUSION: Nanobacteria has a damage effect on HK-2 cells, the damage increases with the acting time expanding. The damage is more severe than that of nHAP. In the damage process of nanobacteria, the lipid peroxidation may play an important role. After the damage of nanobacteria, the adhering effect of the COM crystals to the cells increases observably, and the number of crystals adhering to the cells becomes more and more with the acting time expanding. Although nHAP also has a damage effect on HK-2 cells, it does not effect the adhering process.
Asunto(s)
Bacterias/aislamiento & purificación , Células Epiteliales/patología , Cálculos Renales/microbiología , Túbulos Renales/patología , Animales , Oxalato de Calcio/química , Línea Celular , Cristalización , Humanos , Hidroxiapatitas/química , Cálculos Renales/etiología , Túbulos Renales/microbiología , Nanopartículas , RatasRESUMEN
OBJECTIVE: To understand the relationship between urinary stones and the gut microbiome and to screen for microbial species that may be involved in stone formation. METHODS: Stool samples were collected from patients with urolithiasis and healthy patients between March and December 2017. The samples were analyzed by 16S sequencing to determine differences in the microbiome profiles between the two groups. The mouse model was established and was divided into two groups. Fecal samples were collected from the mice before gavage and three weeks postgavage for microbiome analysis. The microbial population of each group was analyzed to screen for microbial species that may affect the formation of urinary stones. Differences in the number of crystals in the renal tubules of the mice were examined by necropsy. RESULTS: The microbial composition was different between urolithiasis patients and healthy controls. The urolithiasis patients had significantly reduced microbial abundance; however, increased proportions of Bacteroidetes and Actinobacteria were detected compared to healthy controls. Furthermore, the abundance of Alistipesindistinctus and Odoribactersplanchnicus was significantly increased in the urolithiasis patients compared to the healthy controls. In addition, the incidence of urolithiasis was much higher in the experimental mouse group (stone solution + urolithiasis patient stool) than in the control mouse group. However, the microbial abundance before gavage was not significantly different from that seen three weeks postgavage. CONCLUSION: Theurolithiasis patients in this study had a different gut microbiome when compared with that of healthy individuals. The altered microbiome increased the rate of crystal formation in renal tubules and accelerated urinary stone formation in the mouse model of urolithiasis.
Asunto(s)
Microbioma Gastrointestinal/genética , Cálculos Urinarios/microbiología , Actinobacteria/genética , Animales , Bacteroidetes/genética , Heces/microbiología , Femenino , Humanos , Túbulos Renales/microbiología , Masculino , Ratones , Urolitiasis/microbiologíaRESUMEN
Kidney ischemia-reperfusion injury (IRI) is, in part, mediated by immune and inflammatory factors. Since microbial stimuli are known to alter immune and inflammatory responses, we hypothesized that differences in perinatal microbial status would modify renal injury following IRI. We performed bilateral renal IRI on 6-wk-old germ-free and control mice and studied the effects on kidney lymphocyte trafficking, cytokines, function, and structure. Compared with control mice, normal kidneys of germ-free mice exhibited more NKT cells and lower IL-4 levels. Postischemia, more CD8 T cells trafficked into postischemic kidneys of germ-free mice compared with control mice. Renal structural injury and functional decline following IRI were more severe in germ-free mice compared with control mice. When germ-free mice were conventionalized with the addition of bacteria to their diet, the extent of renal injury after IRI became equivalent to age-matched control mice, with similar numbers and phenotypes of T cells and NKT cells, as well as cytokine expression in both normal kidneys and postischemic kidneys of conventionalized germ-free mice and age-matched control mice. Thus microbial stimuli influence the phenotype of renal lymphocytes and the expression of cytokines of normal kidneys and also modulate the outcome of IRI.
Asunto(s)
Enfermedades Renales/microbiología , Enfermedades Renales/patología , Nefritis/microbiología , Nefritis/patología , Daño por Reperfusión/microbiología , Daño por Reperfusión/patología , Animales , Linfocitos T CD8-positivos/microbiología , Linfocitos T CD8-positivos/fisiología , Citocinas/biosíntesis , Citocinas/genética , Vida Libre de Gérmenes , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Interleucina-4/biosíntesis , Intestinos/microbiología , Pruebas de Función Renal , Médula Renal/microbiología , Médula Renal/patología , Túbulos Renales/microbiología , Túbulos Renales/patología , Recuento de Linfocitos , Masculino , Ratones , Monocitos/fisiología , Fenotipo , Linfocitos T/microbiología , Linfocitos T/fisiologíaRESUMEN
Intranuclear and cytoplasmic virus particles of the herpes type were located in epithelial cells that line the kidney collecting tubules obtained from a chick with Marek's disease. The chick had contracted the disease by direct contact transmission. The virus was not observed in any of the invading tumor cells in the same kidney.
Asunto(s)
Herpesviridae/aislamiento & purificación , Enfermedades Renales/microbiología , Túbulos Renales/microbiología , Enfermedades de las Aves de Corral/microbiología , Animales , Pollos , Enfermedades Renales/patología , Enfermedades Renales/veterinaria , Microscopía Electrónica , Enfermedades de las Aves de Corral/patologíaRESUMEN
In dogs with leptospirosis, doxycycline therapy is recommended as the preferred therapy for its ability to eliminate the organism from all tissues, including the renal tubules. Elimination of organisms from the renal tubules terminates leptospiruria and prevents transmission of the organism. This report describes the discovery of persistent leptospiruria in the face of therapy with doxycycline in four dogs and enrofloxacin in one dog. Leptospiruria was confirmed by polymerase chain reaction testing for pathogenic leptospires in all five dogs. In two dogs, leptospiruria resolved after a change in therapy to enrofloxacin. In three dogs, doxycycline and/or enrofloxacin were ineffective at eliminating leptospiruria, which then resolved after therapy with clarithromycin. Pet owners could be at risk as persistent leptospiruria poses a potential zoonotic risk. The potential reasons for persistent leptospiruria as demonstrated by polymerase chain reaction testing are discussed.
Asunto(s)
Antibacterianos/uso terapéutico , Bacteriuria/veterinaria , Enfermedades de los Perros/tratamiento farmacológico , Doxiciclina/uso terapéutico , Enrofloxacina/uso terapéutico , Leptospirosis/veterinaria , Animales , Bacteriuria/tratamiento farmacológico , Bacteriuria/microbiología , Bacteriuria/orina , Enfermedades de los Perros/microbiología , Enfermedades de los Perros/orina , Perros , Túbulos Renales/microbiología , Leptospirosis/tratamiento farmacológico , Leptospirosis/microbiología , Leptospirosis/orina , Estudios RetrospectivosRESUMEN
Diabetics have a higher incidence of urinary tract infection (UTI), are infected with a broader range of uropathogens, and more commonly develop serious UTI sequelae than nondiabetics. To better study UTI in the diabetic host, we created and characterized a murine model of diabetic UTI using the pancreatic islet beta-cell toxin streptozocin in C3H/HeN, C3H/HeJ, and C57BL/6 mouse backgrounds. Intraperitoneal injections of streptozocin were used to initiate diabetes in healthy mouse backgrounds, as defined by consecutive blood glucose levels of >250 mg/dl. UTIs caused by uropathogenic Escherichia coli (UTI89), Klebsiella pneumoniae (TOP52 1721), and Enterococcus faecalis (0852) were studied, and diabetic mice were found to be considerably more susceptible to infection. All three uropathogens produced significantly higher bladder and kidney titers than buffer-treated controls. Uropathogens did not have as large an advantage in the Toll-like receptor 4-defective C3H/HeJ diabetic mouse, arguing that the dramatic increase in colonization seen in C3H/HeN diabetic mice may partially be due to diabetic-induced defects in innate immunity. Competition experiments demonstrated that E. coli had a significant advantage over K. pneumoniae in the bladders of healthy mice and less of an advantage in diabetic bladders. In the kidneys, K. pneumoniae outcompeted E. coli in healthy mice but in diabetic mice E. coli outcompeted K. pneumoniae and caused severe pyelonephritis. Diabetic kidneys contained renal tubules laden with communities of E. coli UTI89 bacteria within an extracellular-matrix material. Diabetic mice also had glucosuria, which may enhance bacterial replication in the urinary tract. These data support that this murine diabetic UTI model is consistent with known characteristics of human diabetic UTI and can provide a powerful tool for dissecting this infection in the multifactorial setting of diabetes.
Asunto(s)
Diabetes Mellitus Experimental , Modelos Animales de Enfermedad , Infecciones por Bacterias Gramnegativas/microbiología , Infecciones por Bacterias Grampositivas/microbiología , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Infecciones Urinarias/microbiología , Animales , Enterococcus faecalis/crecimiento & desarrollo , Escherichia coli/crecimiento & desarrollo , Femenino , Infecciones por Bacterias Gramnegativas/patología , Infecciones por Bacterias Grampositivas/patología , Riñón/microbiología , Túbulos Renales/microbiología , Túbulos Renales/patología , Klebsiella pneumoniae/crecimiento & desarrollo , Ratones , Vejiga Urinaria/microbiologíaRESUMEN
BACKGROUND: Tubulo-interstitial nephritis is the main cause of acute renal injury in leptospirosis. The aim of this study was to evaluate renal tubular function and excretion of solutes in leptospirosis patients during a recent outbreak of leptospirosis in Nan province, Thailand. METHODS: Clinical manifestations were recorded and routine laboratory tests were performed upon admission. Renal tubular functions including tubular reabsorption of phosphate (TRP), fractional excretion of magnesium (FE(Mg)), urinary calcium to creatinine ratio (Uca/cr), urine N-acetyl-beta-D glucosaminidase (NAG) and urine beta(2)-microglobulin were serially monitored during 2 weeks after admission. RESULTS: A total of 20 leptospirosis patients were recruited. Nine (45%) patients had acute renal failure (ARF). Increased urine NAG and beta(2)-microglobulin, which indicate proximal tubular dysfunction, were demonstrated in all 20 (100%) patients. Fifteen (75%) patients had hypermagnesuria, whereas 10 (50%) patients had decreased TRP. Renal magnesium (Mg) and phosphate (P) wasting caused hypomagnesaemia and hypophosphataemia in nine and three patients with ARF, respectively. These abnormal findings significantly improved within 2 weeks after admission. CONCLUSIONS: We conclude that renal Mg and P wasting commonly occur in patients with leptospirosis. The measurement of Mg and P levels in both serum and urine of leptospirosis patients, especially those with ARF, is therefore highly recommended.
Asunto(s)
Túbulos Renales/metabolismo , Túbulos Renales/fisiopatología , Leptospirosis/metabolismo , Leptospirosis/fisiopatología , Magnesio/metabolismo , Acetilglucosaminidasa/orina , Adolescente , Adulto , Anciano , Calcio/orina , Creatinina/orina , Femenino , Humanos , Túbulos Renales/microbiología , Leptospira/patogenicidad , Leptospirosis/complicaciones , Masculino , Persona de Mediana Edad , Nefritis Intersticial/etiología , Nefritis Intersticial/metabolismo , Nefritis Intersticial/fisiopatología , Fosfatos/metabolismo , Estudios Prospectivos , Tailandia , Microglobulina beta-2/orinaRESUMEN
Brucellosis is a multisystem disease that may present with a broad spectrum of clinical manifestations. Until now, no studies have been performed on renal tubular disorders in patients with brucellosis. The present study aims to investigate renal tubular disorders in patients with brucellosis. This prospective case-control study includes a total of 31 brucellosis patients (Group 1) and 30 healthy controls (Group 2) matched for age and sex. Renal tubular functions of children who were diagnosed as having brucellosis in outpatient pediatric clinics were evaluated. First-morning urine samples were collected from Group 1 and Group 2 at the same time. Urea, creatinine, potassium, sodium, and phosphorus were determined in serum and urine by an autoanalyzer. Tubular reabsorption and excretion of urine electrolytes were calculated using the related formulas. Patients with brucellosis had significantly lower levels of tubular reabsorption of phosphorus and serum phosphorus than those of the control group. Furthermore, urine sodium and serum potassium levels and fractionated sodium excretion of brucellosis patients were significantly higher than healthy control group. Estimated glomerular filtration rate was remarkably higher in the patient group (P < 0.001).We concluded that tubular and glomerular functional parameters demonstrate deterioration in patients with brucellosis compared to those in healthy participants.
Asunto(s)
Brucelosis/complicaciones , Enfermedades Renales/etiología , Glomérulos Renales/fisiopatología , Túbulos Renales/fisiopatología , Adolescente , Factores de Edad , Biomarcadores/sangre , Biomarcadores/orina , Brucelosis/diagnóstico , Brucelosis/microbiología , Estudios de Casos y Controles , Niño , Femenino , Humanos , Enfermedades Renales/diagnóstico , Enfermedades Renales/microbiología , Enfermedades Renales/fisiopatología , Glomérulos Renales/microbiología , Glomérulos Renales/patología , Túbulos Renales/microbiología , Túbulos Renales/patología , Masculino , Pronóstico , Estudios Prospectivos , Factores de Riesgo , TurquíaRESUMEN
BACKGROUND: Infectious Leptospira colonize the kidneys of reservoir (e.g. rats) and accidental hosts such as humans. The renal response to persistent leptospiral colonization, as measured by urinary protein biosignatures, has not been systematically studied. Urinary exosomes--bioactive membrane-bound nanovesicles--contain cell-state specific cargo that additively reflect formation all along the nephron. We hypothesized that Leptospira-infection will alter the content of urine exosomes, and further, that these Leptospira-induced alterations will hold clues to unravel novel pathways related to bacterial-host interactions. METHODOLOGY/PRINCIPAL FINDINGS: Exosome protein content from 24 hour urine samples of Leptospira-infected rats was compared with that of uninfected rats using SDS-PAGE and liquid chromatography/tandem mass spectrometry (LC-MS/MS). Statistical models were used to identify significantly dysregulated proteins in Leptospira-infected and uninfected rat urine exosomes. In all, 842 proteins were identified by LC-MS/MS proteomics of total rat urine and 204 proteins associated specifically with exosomes. Multivariate analysis showed that 25 proteins significantly discriminated between uninfected control and infected rats. Alanyl (membrane) aminopeptidase, also known as CD13 topped this list with the highest score, a finding we validated by Western immunoblotting. Whole urine analysis showed Tamm-Horsfall protein level reduction in the infected rat urine. Total urine and exosome proteins were significantly different in male vs. female infected rats. CONCLUSIONS: We identified exosome-associated renal tubule-specific responses to Leptospira infection in a rat chronic colonization model. Quantitative differences in infected male and female rat urine exosome proteins vs. uninfected controls suggest that urine exosome analysis identifies important differences in kidney function that may be of clinical and pathological significance.
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Exosomas/metabolismo , Túbulos Renales/inmunología , Túbulos Renales/microbiología , Leptospirosis/inmunología , Proteinuria/metabolismo , Animales , Western Blotting , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Femenino , Interacciones Huésped-Patógeno , Masculino , Modelos Estadísticos , Análisis Multivariante , Proteómica/métodos , Ratas , Factores Sexuales , Espectrometría de Masas en TándemRESUMEN
Evidence suggests that human cytomegalovirus is resident in the kidneys of seropositive donors at the time of transplantation, and CMV has been implicated in both glomerulonephritis and interstitial nephritis. In this study we assessed the interactions of CMV with two human renal cell types in culture: glomerular visceral epithelial cells (GVE) and renal tubular epithelial (RTE) cells. GVE permitted viral adsorption, penetration, nuclear translocation, and restricted viral transcription. However, early viral protein expression was not detectable by immunofluorescence and infectious virions were not produced. In contrast, retinoic acid-treated GVE permitted early viral protein expression and supported CMV replication. RTE also permitted viral adsorption and penetration. CMV-specific early proteins were readily observed by immunofluorescence, and CMV DNA replication was observed by DNA dot blot hybridization. Assays comparing viral yield with viral DNA synthesis indicated that RTE were capable of supporting persistent and prolonged viral expression without significant cell death for at least 55 days after infection. We believe that these findings should explain chronic viruria in individuals with symptomatic and asymptomatic CMV infection. In addition, GVE could also be a potential source of CMV transmission when altered by disease or transplantation.
Asunto(s)
Infecciones por Citomegalovirus/microbiología , Citomegalovirus/crecimiento & desarrollo , Glomérulos Renales/microbiología , Túbulos Renales/microbiología , Células Cultivadas , ADN Viral/análisis , Células Epiteliales , Epitelio/microbiología , Técnica del Anticuerpo Fluorescente , Humanos , Glomérulos Renales/citología , Túbulos Renales/citología , Hibridación de Ácido Nucleico , Factores de Tiempo , Replicación ViralRESUMEN
A primary culture system for the cells of mouse renal-tubular epithelium was established and used to observe the adhesion of leptospires. Virulent strains of serovars copenhageni and ballum attached themselves to epithelial cells within 3 h of infection whereas an avirulent variant of serovar copenhageni did not adhere to epithelial cells at all within the experimental period of 24 h. The saprophytic Leptospira biflexa serovar patoc became attached non-specifically to inert glass surfaces as well as to the cells. The adhesion of leptospires to epithelial cells was not inhibited by homologous antibody.
Asunto(s)
Túbulos Renales/microbiología , Leptospira/crecimiento & desarrollo , Adhesividad , Animales , Anticuerpos Antibacterianos , Reacciones Antígeno-Anticuerpo , Células Cultivadas , Epitelio/microbiología , Técnica del Anticuerpo Fluorescente , Leptospira/inmunología , Leptospira/patogenicidad , Ratones , Microscopía Electrónica de RastreoRESUMEN
Commercial turkeys from four Iowa flocks, two Illinois flocks, and three California flocks were submitted to state diagnostic laboratories because of a variety of health problems. The turkeys ranged in age from 5 to 12 weeks, included both hens and toms, and were owned by five different companies. Some flocks had previously been immunized with live hemorrhagic enteritis vaccine, and other flocks were unvaccinated. In all accessions, basophilic intranuclear inclusion bodies were observed in renal tubular epithelium by light microscopy. Transmission electron microscopy showed that the inclusions consisted of densely packed virus particles. The virions were identified as adenoviruses based upon the icosahedral morphology and average particle diameters of 72 nm. Avidin-biotin immunoperoxidase staining of formalin-fixed, paraffin-embedded kidneys was used to identify this adenovirus as hemorrhagic enteritis virus.
Asunto(s)
Infecciones por Adenoviridae/veterinaria , Aviadenovirus , Cuerpos de Inclusión Viral/patología , Enfermedades Renales/veterinaria , Enfermedades de las Aves de Corral/patología , Pavos/microbiología , Infecciones por Adenoviridae/patología , Animales , Aviadenovirus/aislamiento & purificación , Células Epiteliales , Femenino , Técnicas para Inmunoenzimas/veterinaria , Enfermedades Renales/microbiología , Enfermedades Renales/patología , Túbulos Renales/microbiología , Masculino , Microscopía Electrónica/veterinaria , Enfermedades de las Aves de Corral/microbiologíaRESUMEN
A retrospective study was conducted to evaluate the temporal relationship between flock seroconversion to hemorrhagic enteritis virus (HEV) and the appearance of adenoviral inclusions in the spleen and renal tubular epithelium. The study was conducted on samples of turkey poults submitted to the Fresno Branch of the California Veterinary Diagnostic Laboratory System during May to December 1988. The study included 78 submissions (four to eight poults per submission) of ages ranging from 6 to 15 weeks. Sera were tested for antibodies to HEV using the agar gel immunodiffusion test. Spleen and kidney samples were examined by light microscopy for the presence of inclusions in the mononuclear phagocytes of the spleen or in the renal tubular epithelium of the kidney. Logistic regression statistical analysis was used to evaluate the association between the age of the bird and the likelihood of the presence of inclusions in the spleen and kidney, as well as the likelihood of seroconversion to HEV. A significant association (P less than 0.05) was found between the presence of splenic inclusion bodies and the age of the bird. The probability of splenic inclusions was higher in younger birds (6 weeks of age), and decreased as the birds became older, approaching zero at 11 weeks of age. The kidney inclusions were significantly associated with age. The probability of detecting the inclusions increased with age, reached a maximum at 10 weeks, and then declined, approaching zero by 14 weeks. However, the probability of seroconversion to HEV increased significantly with age up to 10 weeks and then remained positive throughout the remainder of the study period.
Asunto(s)
Infecciones por Adenoviridae/veterinaria , Aviadenovirus/aislamiento & purificación , Enteritis/veterinaria , Enfermedades de las Aves de Corral/microbiología , Pavos , Infecciones por Adenoviridae/inmunología , Infecciones por Adenoviridae/microbiología , Factores de Edad , Animales , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/sangre , Aviadenovirus/inmunología , Aviadenovirus/ultraestructura , Enteritis/inmunología , Enteritis/microbiología , Epitelio/microbiología , Hemorragia Gastrointestinal/inmunología , Hemorragia Gastrointestinal/microbiología , Hemorragia Gastrointestinal/veterinaria , Cuerpos de Inclusión Viral/ultraestructura , Túbulos Renales/microbiología , Microscopía Electrónica , Enfermedades de las Aves de Corral/inmunología , Estudios Retrospectivos , Bazo/microbiología , Virión/ultraestructuraRESUMEN
Glycolipoprotein (GLP) cytotoxin was extracted from Leptospira interrogans serovar canicola. The silver staining profile of GLP subjected to SDS-PAGE under denaturing conditions showed a number of bands in the mol. weight range of 14-66 kDa. Mouse Monoclonal Antibodies (MAbs) IgG3 recognizing a band near to 24 kDa of leptospiral GLP were produced (clone number MGLP-01). The agglutinating property of MAbs was established by microscopic agglutination test (MAT) using 25 different serovars as antigens. Only the homologous serovar was agglutinated by MAbs suggesting that the recognized epitope is a specific surface-exposed antigen. The MAbs were applied to demonstration of leptospiral antigens in tissue damage by avidin-biotin immunoperoxidase staining. Golden hamsters were experimentally infected with a virulent strain of L. interrogans serovar canicola. Histologically kidneys stained by routine hematoxylin and eosin showed changes characterized by injury of tubular epithelial cells leading to acute tubular necrosis (ATN). Typical, well-defined morphologic leptospires or finely granular deposits were found by immunoperoxidase staining near to blood vessels, within inflammatory infiltrates and intraluminal in proximal and distal parts of the nephron. Binding of leptospiral antigens to capillary endothelial cells, tubular epithelial cells and macrophages were also demonstrated. This entails a basis for further studies either in research or in diagnostic histopathology.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/análisis , Necrosis Tubular Aguda/microbiología , Leptospira interrogans/inmunología , Enfermedad de Weil/microbiología , Animales , Proteínas Bacterianas/aislamiento & purificación , Cricetinae , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Humanos , Hibridomas , Técnicas para Inmunoenzimas , Necrosis Tubular Aguda/patología , Túbulos Renales/microbiología , Mesocricetus , Ratones , Ratones Endogámicos C57BL , Enfermedad de Weil/patologíaRESUMEN
Kidney tissues were removed from euthanatized mature White Leghorn chickens 4 days after IV inoculation with type A influenza virus. The kidney tissues were then fixed at -70 C, using a freeze substitution technique. Type A influenza virus nucleoprotein was readily detected in the nuclei and cytoplasm of the proximal and distal tubular epithelial cells by immunocytochemistry, and the sharpness of the immunomarker in the cells indicated minimal antigen migration during fixation and tissue section preparation. This tissue fixation technique also resulted in good preservation of cellular morphology. The freeze substitution technique of tissue fixation is an excellent alternative to cryostat-cut acetone-fixed tissue sections or conventional chemical fixation of paraffin-embedded tissues for in situ immunocytochemical localization of type A influenza virus nucleoprotein antigen.
Asunto(s)
Virus de la Influenza A/aislamiento & purificación , Riñón/microbiología , Nucleoproteínas/análisis , Animales , Embrión de Pollo , Pollos , Epitelio/microbiología , Congelación , Inmunohistoquímica , Túbulos Renales/microbiología , MasculinoRESUMEN
Histological examination of 69 pairs of infected kidneys from 12 species of Rodentia, Lagomorpha, Carnivora and Insectivora revealed that leptospires were confined mainly to the proximal and distal convoluted tubule, were less often found in the thick loop of Henle and only rarely in the collecting duct. On no occasion were the organisms present in the thin loop of Henle. Preliminary observations on the relationship of leptospires to tubule epithelium indicate some degree of physical attachment. It is suggested that the avoidance of the thin loop of Henle might be a reflection of its structural properties.