RESUMEN
BACKGROUND: To protect the kidney effectively with medication in type 2 diabetics, it is crucial to identify such at-risk patients early for treatment. We investigated whether peptiduria precedes proteinuria (the earliest urinary marker in our model), and thereby serve as an early predictor of diabetic nephropathy. METHODS: A longitudinal study was performed in a rat model of diabetic nephropathy. Peptides, defined as degradation products of proteins of < 13 kD size, were quantified by a previously validated method using a combination of Lowry and Biorad protein assays. Peptides in urine were also confirmed by chromatographically separating low molecular weight fractions from urine and quantifying albumin fragments in these fractions by enzyme immunoassay. Also, the mechanism of peptiduria was addressed by measuring acid phosphatase, a marker of lysosomal activity, in urine and on kidney sections (histochemically). RESULTS: In rats with diabetic nephropathy, proteinuria occurred after 12 weeks of diabetes, while peptiduria occurred as early as 2 weeks after diabetes. Peptiduria was confirmed by showing that the chromatographically separated low molecular weight fractions of urine containing albumin fragments is in proportion to the level of peptiduria. The time course of peptiduria paralleled the increase in urinary acid phosphatase suggesting that the mechanism of early peptiduria could be due to upregulation of lysosomal enzyme activity in the tubules. CONCLUSIONS: Our results showing that peptiduria precedes proteinuria in diabetic nephropathy provide a compelling rationale to perform a prospective human clinical trial to investigate whether peptiduria can serve as an early predictor of diabetic nephropathy.
Asunto(s)
Nefropatías Diabéticas/diagnóstico , Nefropatías Diabéticas/orina , Péptidos/orina , Fosfatasa Ácida/orina , Albuminuria/orina , Animales , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/orina , Estudios Longitudinales , Lisosomas/enzimología , Masculino , Peso Molecular , Valor Predictivo de las Pruebas , Proteinuria/etiología , Proteinuria/orina , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Previously we found that kidney tissue and urinary exosomes from patients of diabetic kidney disease showed high levels of ceruloplasmin (CP). Because CP is an acute-phase protein of kidney origin, it could be an early marker of many other kidney diseases. To investigate this hypothesis, we first measured urine exosomal and kidney expression of CP in non-diabetic chronic kidney disease (CKD) patients (membranous nephropathy, focal segmental glomerulosclerosis, lupus nephritis and IgA nephropathy) followed by a longitudinal study in rat passive Heymann nephritis (PHN), a model of human membranous nephropathy. METHODS: Urinary exosomes were isolated from urine of patients (and rats) by differential centrifugation. The exosomal extracts were used for measuring CP using ELISA. Kidney expression of CP was evaluated by immune-staining biopsy tissues. Similar techniques were applied in rat PHN model (produced by injection of anti-gp600 antiserum) to analyze urine exosomal and kidney CP. RESULTS: Urine exosomal CP levels were 10-20 times higher in CKD patients than in controls; consistent with this we found high immune-reactive CP localized in tubules and collecting ducts of biopsies of CKD patients. In the PHN model urinary exosomal CP level was significantly higher prior to the onset of proteinuria. Early rise of urine exosomal CP, which preceded proteinuria, correlated with high immunoreactive CP found in rat kidneys at this time. CONCLUSION: We propose that urine exosomal CP, observed to increase prior to proteinuria, makes it a potential urinary biomarker to diagnose early kidney disease.
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Ceruloplasmina/orina , Exosomas/enzimología , Glomerulonefritis Membranosa/orina , Riñón/enzimología , Proteinuria/orina , Insuficiencia Renal Crónica/orina , Adulto , Animales , Biomarcadores/orina , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Diagnóstico Precoz , Exosomas/patología , Femenino , Glomerulonefritis Membranosa/diagnóstico , Glomerulonefritis Membranosa/enzimología , Humanos , Riñón/patología , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Proteinuria/diagnóstico , Proteinuria/enzimología , Ratas Sprague-Dawley , Insuficiencia Renal Crónica/diagnóstico , Insuficiencia Renal Crónica/enzimología , Regulación hacia ArribaRESUMEN
Intravascular hemolysis and hemoglobinuria are associated with sickle cell nephropathy. ApoL1 is involved in cell-free hemoglobin scavenging through association with haptoglobin-related protein. APOL1 G1/G2 variants are the strongest genetic predictors of kidney disease in the general African-American population. A single report associated APOL1 G1/G2 with sickle cell nephropathy. In 221 patients with sickle cell disease at the University of Illinois at Chicago, we replicated the finding of an association of APOL1 G1/G2 with proteinuria, specifically with urine albumin concentration (ß=1.1, P=0.003), observed an even stronger association with hemoglobinuria (OR=2.5, P=4.3×10(-6)), and also replicated the finding of an association with hemoglobinuria in 487 patients from the Walk-Treatment of Pulmonary Hypertension and Sickle cell Disease with Sildenafil Therapy study (OR=2.6, P=0.003). In 25 University of Illinois sickle cell disease patients, concentrations of urine kidney injury molecule-1 correlated with urine cell-free hemoglobin concentrations (r=0.59, P=0.002). Exposing human proximal tubular cells to increasing cell-free hemoglobin led to increasing concentrations of supernatant kidney injury molecule-1 (P=0.01), reduced viability (P=0.01) and induction of HMOX1 and SOD2. HMOX1 rs743811 associated with chronic kidney disease stage (OR=3.0, P=0.0001) in the University of Illinois cohort and end-stage renal disease (OR=10.0, P=0.0003) in the Walk-Treatment of Pulmonary Hypertension and Sickle cell Disease with Sildenafil Therapy cohort. Longer HMOX1 GT-tandem repeats (>25) were associated with lower estimated glomerular filtration rate in the University of Illinois cohort (P=0.01). Our findings point to an association of APOL1 G1/G2 with kidney disease in sickle cell disease, possibly through increased risk of hemoglobinuria, and associations of HMOX1 variants with kidney disease, possibly through reduced protection of the kidney from hemoglobin-mediated toxicity.
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Anemia de Células Falciformes/complicaciones , Anemia de Células Falciformes/genética , Variación Genética , Hemoglobinas/genética , Hemoglobinas/metabolismo , Enfermedades Renales/etiología , Enfermedades Renales/metabolismo , Adulto , Apolipoproteína L1 , Apolipoproteínas/genética , Línea Celular , Estudios de Cohortes , Femenino , Expresión Génica , Predisposición Genética a la Enfermedad , Genotipo , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Hemoglobinuria , Receptor Celular 1 del Virus de la Hepatitis A , Humanos , Túbulos Renales Proximales/metabolismo , Lipoproteínas HDL/genética , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/orina , Persona de Mediana Edad , Receptores Virales/genéticaRESUMEN
BACKGROUND: Predicting or diagnosing underlying kidney disease by analyzing whole urine remains the mainstay of nephrology practice. However, whole urine is a poor compartment to assess many structural changes in the kidney because whole urine contains only a few proteins derived from the kidney itself. Urinary exosomes, on the other hand, which are derived from the kidney, contain proteins secreted by the kidney. We experimentally tested the hypothesis that 'urinary exosomes more faithfully represent changes in the kidney tissue than whole urine'. A direct comparison between whole urine and urine exosomal levels of two chosen kidney disease markers, gelatinase and ceruloplasmin, was carried out on diabetic kidney disease patients. METHODS: Urinary exosomes were separated from whole urine by sequential centrifugation including ultra-centrifugation. Gelatinase activity was measured using fluorosceinated gelatin as the substrate, and ceruloplasmin was measured by sandwich ELISA. A few kidney specimens from patients biopsied for atypical features were histochemically stained for validation of the biochemical results. RESULTS: We found that changes in both, gelatinase (decreased activity) and ceruloplasmin (increased levels), in the urinary exosomes of diabetic kidney patients were in agreement with the alterations of these two proteins in the kidney tissue. In contrast, the levels of these two proteins in whole urine were highly variable and did not correlate with levels in the diabetic kidney tissue. CONCLUSION: In conclusion, these results confirmed our hypothesis that protein markers in urinary exosomes better reflected the underlying protein changes in the kidney than in whole urine samples.
Asunto(s)
Ceruloplasmina/orina , Nefropatías Diabéticas/diagnóstico , Nefropatías Diabéticas/orina , Exosomas/química , Gelatinasas/orina , Adulto , Albúminas/química , Biomarcadores/orina , Biopsia , Creatinina/orina , Diabetes Mellitus Tipo 2/orina , Ensayo de Inmunoadsorción Enzimática , Femenino , Fluoresceína/química , Humanos , Masculino , Persona de Mediana Edad , UltracentrifugaciónRESUMEN
Stem cells show promise in the treatment of AKI but do not survive long term after injection. However, organ repair has been achieved by extending and attaching the omentum, a fatty tissue lying above the stomach containing stem cells, to various organs. To examine whether fusing the omentum to a subtotally nephrectomized kidney could slow the progression of CKD, we used two groups of rats: an experimental group undergoing 5/6 nephrectomy only and a control group undergoing 5/6 nephrectomy and complete omentectomy. Polydextran gel particles were administered intraperitoneally before suture only in the experimental group to facilitate the fusion of the omentum to the injured kidney. After 12 weeks, experimental rats exhibited omentum fused to the remnant kidney and had lower plasma creatinine and urea nitrogen levels; less glomerulosclerosis, tubulointerstitial injury, and extracellular matrix; and reduced thickening of basement membranes compared with controls. A fusion zone formed between the injured kidney and the omentum contained abundant stem cells expressing stem cell antigen-1, Wilms' tumor 1 (WT-1), and CD34, suggesting active, healing tissue. Furthermore, kidney extracts from experimental rats showed increases in expression levels of growth factors involved in renal repair, the number of proliferating cells, especially at the injured edge, the number of WT-1-positive cells in the glomeruli, and WT-1 gene expression. These results suggest that contact between the omentum and injured kidney slows the progression of CKD in the remnant organ, and this effect appears to be mediated by the presence of omental stem cells and their secretory products.
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Células Madre Adultas/fisiología , Glomeruloesclerosis Focal y Segmentaria/fisiopatología , Epiplón/fisiología , Insuficiencia Renal Crónica/fisiopatología , Tejido Adiposo/citología , Tejido Adiposo/fisiología , Tejido Adiposo/cirugía , Células Madre Adultas/citología , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Mesangio Glomerular/metabolismo , Mesangio Glomerular/patología , Mesangio Glomerular/fisiopatología , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomeruloesclerosis Focal y Segmentaria/patología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Nefrectomía , Epiplón/citología , Epiplón/cirugía , Comunicación Paracrina/fisiología , Ratas , Ratas Sprague-Dawley , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patologíaRESUMEN
BACKGROUND: In previous studies, we obtained mesenchymal stem cells called granulation tissue stem cells (GTSC) from a regenerating granulation tissue created by placing a foreign body in the subcutaneous tissue of rats. Here, we used GTSC to ameliorate ischemia/reperfusion-induced acute kidney injury (AKI) in rats. METHODS: In two groups of Fischer rats, we induced ischemia/reperfusion injury. Group 1 (treated rats) received one intravenous injection of GTSC 3 h after injury; Group 2 (control rats) received vehicle. Both groups were subsequently studied by renal function tests, kidney histology and immunohistochemistry. RESULTS: At 24 and 48 h after injury, plasma creatinine and blood urea nitrogen were significantly lower in the treated rats as compared to control rats. The levels remained low and declined to near baseline levels by Day 4 in the treated group. At the cortico-medullary region, the treated rats showed significantly higher renal tubular cell proliferation and less tubular cell apoptosis. Histological analysis of the kidney for tubular dilatation, necrosis, congestion and casts was not significantly different in the two groups. To understand the mechanism of the GTSC effect, messenger RNA levels of several growth and immune modulatory factors were quantified in cultured GTSC and compared with those in cultured glomerular epithelial cell (GEC; a non-stem cell line). GTSC had 2- to 8-fold higher expression of FGF2, HGF, IGF-1, vascular endothelial growth factor (growth factors) and IL-4, IL-6 (anti-inflammatory factors) than GEC. CONCLUSIONS: Administration of GTSC accelerates recovery in rats with ischemia/reperfusion-induced AKI. This effect may be mediated by the paracrine action of growth and immune-suppressive factors secreted by these cells.
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Lesión Renal Aguda/tratamiento farmacológico , Lesión Renal Aguda/etiología , Cuerpos Extraños , Tejido de Granulación/patología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Daño por Reperfusión/complicaciones , Lesión Renal Aguda/metabolismo , Animales , Apoptosis , Proliferación Celular , Células Cultivadas , Creatinina/sangre , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Interleucina-4/metabolismo , Interleucina-6/metabolismo , Túbulos Renales/metabolismo , Túbulos Renales/patología , Masculino , Células Madre Mesenquimatosas/metabolismo , Modelos Animales , Ratas , Ratas Endogámicas F344 , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Homozygosity for the hemoglobin (Hb) S mutation (HbSS, sickle cell anemia) results in hemoglobin polymerization under hypoxic conditions leading to vaso-occlusion and hemolysis. Sickle cell anemia affects 1:500 African Americans and is a strong risk factor for kidney disease, although the mechanisms are not well understood. Heterozygous inheritance (HbAS; sickle cell trait) affects 1:10 African Americans and is associated with an increased risk for kidney disease in some reports. Using transgenic sickle mice, we investigated the histopathologic, ultrastructural, and gene expression differences with the HbS mutation. Consistent with progressive glomerular damage, we observed progressively greater urine protein concentrations (P = 0.03), glomerular hypertrophy (P = 0.002), and glomerular cellularity (P = 0.01) in HbAA, HbAS, and HbSS mice, respectively. Ultrastructural studies demonstrated progressive podocyte foot process effacement, glomerular basement membrane thickening with reduplication, and tubular villous atrophy with the HbS mutation. Gene expression studies highlighted the differential expression of several genes involved in prostaglandin metabolism (AKR1C18), heme and iron metabolism (HbA-A2, HMOX1, SCL25A37), electrolyte balance (SLC4A1, AQP6), immunity (RSAD2, C3, UBE2O), fatty acid metabolism (FASN), hypoxia hall-mark genes (GCK, SDC3, VEGFA, ETS1, CP, BCL2), as well as genes implicated in other forms of kidney disease (PODXL, ELMO1, FRMD3, MYH9, APOA1). Pathway analysis highlighted increased gene enrichment in focal adhesion, extracellular matrix-receptor interaction, and axon guidance pathways. In summary, using transgenic sickle mice, we observed that inheritance of the HbS mutation is associated with glomerular and tubular damage and identified several candidate genes and pathways for future investigation in sickle cell trait and sickle cell anemia-related kidney disease.
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Anemia de Células Falciformes/patología , Progresión de la Enfermedad , Glomérulos Renales/patología , Túbulos Renales/patología , Rasgo Drepanocítico/patología , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/genética , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Hipertrofia , Glomérulos Renales/ultraestructura , Túbulos Renales/ultraestructura , Ratones Transgénicos , Rasgo Drepanocítico/sangre , Rasgo Drepanocítico/genéticaRESUMEN
We describe a case of lipoprotein glomerulopathy, the second ever reported from the United States, in a Mexican man with a hitherto undescribed mutation in the apolipoprotein E gene (substitution of proline for arginine at position 147 [Arg147Pro]). In this patient, glomerular basement membranes showed double contours and circumferential mesangial extensions, suggesting that deposition of lipids could be injurious to endothelial cells. Immunofluorescence staining of thrombi was positive for apolipoprotein E and B. To study the reason for lipid deposition in glomeruli, we incubated normal human kidney sections with serum from the patient and a healthy control. Apolipoprotein E from the patient's serum showed binding to the glomerular capillary wall, but the control did not, suggesting enhanced binding of the mutated apolipoprotein E to glomerular capillaries. Apolipoprotein E genotyping by means of restriction endonuclease digestion of polymerase chain reaction-amplified genomic DNA showed it to be of the wild-type E3/E3.
Asunto(s)
Apolipoproteínas E/genética , Glomérulos Renales/irrigación sanguínea , Lipoproteínas , Mutación , Trombosis/genética , Adulto , Apolipoproteínas E/metabolismo , Capilares , Humanos , Glomérulos Renales/metabolismo , Masculino , LinajeRESUMEN
The role of the Na(+)-coupled HCO(3)(-) transporter (NBC) family is indispensable in acid-base homeostasis. Almost all tissues express a member of the NBC family. NBC has been studied extensively in the kidney and plays a role in proximal tubule HCO(3)(-) reabsorption. Although the exact function of this transporter family on other tissues is not very clear, the ubiquitous expression of NBC family suggests a role in cell pH regulation. Altered NBC activity caused by mutations of the gene responsible for NBC protein expression results in pathophysiologic conditions. Mutations of NBC resulting in important clinical disorders have been reported extensively on one member of the NBC family, the kidney NBC (NBC1). These mutations have led to several structural studies to understand the mechanism of the abnormal NBC1 activity.
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Riñón/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Acidosis Tubular Renal/genética , Acidosis Tubular Renal/metabolismo , Animales , Humanos , Simportadores de Sodio-Bicarbonato/química , Simportadores de Sodio-Bicarbonato/genéticaRESUMEN
AIM: To investigate whether we could create natural autologous tissue patches in the subcutaneous space for organ repair. METHODS: We implanted the following three types of inert foreign bodies in the subcutaneous tissue of rats to produce autologous tissue patches of different geometries: (1) a large-sized polyvinyl tube (L = 25 mm, internal diameter = 7 mm) sealed at both ends by heat application for obtaining a large flat piece of tissue patch for organ repair; (2) a fine polyvinyl tubing (L = 25 mm, internal diameter = 3 mm) for creating cylindrically shaped grafts for vascular or nerve repair; and (3) a slurry of polydextran particle gel for inducing a bladder-like tissue. Implantation of inert materials was carried out by making a small incision on one or either side of the thoracic-lumbar region of rats. Subcutaneous pockets were created by blunt dissection around the incision into which the inert bodies were inserted (1 or 2 per rat). The incisions were closed with silk sutures, and the animals were allowed to recover. In case of the polydextran gel slurry 5 mL of the slurry was injected in the subcutaneous space using an 18 gauge needle. After implanting the foreign bodies a newly regenerated encapsulating tissue developed around the foreign bodies. The tissues were harvested after 4-42 d of implantation and studied by gross examination, histology, and histochemistry for organization, vascularity, and presence of mesenchymal stem cells (MSCs) (CD271+CD34+ cells). RESULTS: Implanting a large cylindrically shaped polyvinyl tube resulted in a large flat sheet of tissue that could be tailored to a specific size and shape for use as a tissue patch for repairing large organs. Implanting a smaller sized polyvinyl tube yielded a cylindrical tissue that could be useful for repairing nerves and blood vessels. This type of patch could be obtained in different lengths by varying the length of the implanted tube. Implanting a suspension of inert polydextran suspension gave rise to a bladder-like tissue that could be potentially used for repairing heart valves. Histologically, the three different types of tissue patches generated were organized similarly, consisting of three layers, increasing in thickness until day 14. The inner layer in contact with the inert material was avascular; a middle layer that was highly vascular and filled with matrix, and an outer layer consisting of loose connective tissue. MSCs identified as CD271+CD34+ cells were present in the medial layer and around major blood vessels at day 4 but absent at later time points. The early-harvested tissues, endowed with MSCs, could be used for tissue repair, while the later-harvested tissues, being less vascular but thicker and tougher, could be used as filler tissue for cosmetic purposes. CONCLUSION: An autologous, vascularized tissue patch of desired shape and size can be created in the subcutaneous space by implanting different types of inert bodies.
RESUMEN
BACKGROUND: Normal urine contains low-molecular-weight peptides or protein fragments that have been poorly studied, primarily because of the technical difficulty of measuring peptides in the presence of proteins. We studied these substances in healthy subjects and patients with renal disease and varying degrees of proteinuria to understand the factors that determine their excretion. METHODS: We estimated these substances as the difference between results using the Lowry method (which detects both proteins and peptides) and those obtained using the dye-binding Bradford (Biorad) method (Biorad Laboratories Inc, Hercules, CA; which detects only proteins). RESULTS: We validated this 2-assay approach to measure peptide levels by showing that such proteins as immunoglobulin G, albumin, and lysozyme were measured equally by the Lowry and Biorad methods, whereas degraded proteins were recognized by the Lowry method only, but not by the Biorad method. We found that healthy subjects excreted less than 200 mg of protein, but 3 to 4 g of peptides/g creatinine; thus, peptides constituted approximately 95% of total protein material excreted in urine. Patients with renal disease and proteinuria had a progressive decrease in peptide excretion, ranging from 3 to 0 g/g creatinine. Twenty-five percent of nephrotic patients (18 of 72 patients) excreted very small amounts of peptides in urine (0% to 10% of total protein material). CONCLUSION: We found that healthy persons excrete substantial amounts of peptides in urine, and this excretion decreases in the presence of proteinuric renal disease. It is possible that these peptides in urine arise from the tubular degradation of filtered proteins and exocytosis of protein fragments toward the urinary side, a process that becomes increasingly impaired as proteinuria increases.
Asunto(s)
Enfermedades Renales/orina , Péptidos/orina , HumanosRESUMEN
IMPORTANCE: Elevated intraocular pressure (IOP) and decreased ocular perfusion pressure (OPP) are risk factors for glaucoma development and progression. Unrecognized significant IOP elevation or OPP reduction during hemodialysis (HD) could lead to glaucomatous optic nerve damage and subsequent visual loss. OBJECTIVE: To evaluate changes in IOP and OPP during HD. DESIGN, SETTING, AND PARTICIPANTS: A cross-sectional observational study was conducted in patients undergoing HD at an ambulatory care clinic at the University of Illinois at Chicago. EXPOSURES: Forty-nine patients (97 eyes) undergoing HD were enrolled. Exclusion criteria included preexisting corneal abnormalities, history of corneal surgery, allergy to topical anesthetic agents, and current eye infection. Nine patients had previous diagnoses of open-angle glaucoma (OAG) or suspected glaucoma. At 3 time points, IOP was measured using a pneumatonometer and blood pressure was recorded. Measurements were made with the patient in a seated position approximately 15 minutes before starting HD (T1), approximately 2 hours after starting HD (T2), and approximately 15 minutes after ending HD (T3). Mean arterial pressure (MAP) and OPP (systolic, diastolic, and mean OPP) were calculated. MAIN OUTCOMES AND MEASURES: Intraocular pressure and OPP. RESULTS: From T1 to T3, IOP significantly increased by 3.1 mm Hg (both eyes, P < .001), MAP significantly decreased by 5.8 mm Hg (P = .05), and all OPP measures significantly decreased from baseline (all P ≤ .02). Using previously reported thresholds of increased glaucoma development and progression risk, 53% of the right eyes (26 of 49) and 46% of the left eyes (22 of 48) had a systolic OPP of 101 mm Hg or less, 71% of the right eyes (35 of 49) and 73% of the left eyes (35 of 48) had a diastolic OPP of 55 mm Hg or less, and 63% of the right eyes (31 of 49) and 65% of the left eyes (31 of 48) had a mean OPP of 42 mm Hg or less. CONCLUSIONS AND RELEVANCE: Significantly increased IOP and decreased OPP occur during HD, bringing both to levels that increase the risk of glaucoma development and progression. Clinicians should consider HD history in patients who have glaucoma progression, even when IOP has been well controlled. Such patients may benefit from IOP and blood pressure monitoring during HD sessions to minimize OPP changes resulting from IOP spikes and/or suboptimal blood pressure.
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Presión Arterial/fisiología , Glaucoma de Ángulo Abierto/fisiopatología , Presión Intraocular/fisiología , Fallo Renal Crónico/fisiopatología , Hipertensión Ocular/fisiopatología , Diálisis Renal , Adulto , Anciano , Anciano de 80 o más Años , Estudios Transversales , Femenino , Humanos , Fallo Renal Crónico/terapia , Masculino , Persona de Mediana Edad , Postura , Estudios Prospectivos , Esfigmomanometros , Tonometría OcularRESUMEN
Earlier we showed that when omentum, activated by inert particles, is allowed to fuse to a wedge cut in the liver, it induces stem cell proliferation in the liver resulting in massive liver regeneration. Here, we attempt to culture stem cells from the omentum-induced regenerating liver tissue. Cells from regenerating liver tissue were harvested and cultured. Cultured cells were characterized by immune staining, fluorescence activated cell sorting analysis, growth factor assay, in vitro differentiation, and their ability to engraft to injured sites in vivo. Culture yielded cells with a mesenchymal stem cell phenotype that could be maintained in culture indefinitely. These cells, called regenerating liver stem cells, expressed both adult and embryonic stem cell markers, secreted high levels of vascular endothelial growth factor, and expressed albumin. When grown on matrigel in the presence of hepatocyte growth factor, these cells differentiated into hepatocyte-like cells in culture, but they did not differentiate to adipogenic and osteogenic lineages when grown in specific differentiation medium. The differentiated cells expressed α-fetoprotein and secreted high levels of albumin and urea. After systemic injection, the undifferentiated cells engrafted only to the injured sites in the liver and not to the normal areas of the liver. In conclusion, omentum-induced regenerating liver yields hepatocyte-committed stem cells in culture. Such cells could prove to be useful in cell transplantation therapies.
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Hepatocitos/citología , Regeneración Hepática/fisiología , Hígado/lesiones , Epiplón/fisiología , Células Madre/fisiología , Adulto , Animales , Técnicas de Cultivo de Célula/métodos , Trasplante de Células/métodos , Citometría de Flujo , Factor de Crecimiento de Hepatocito/fisiología , Hepatocitos/fisiología , Humanos , Neoplasias Renales/genética , Neoplasias Renales/patología , Neoplasias Renales/fisiopatología , Hígado/citología , Hígado/fisiología , Masculino , Epiplón/citología , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Células Madre/citología , Supresión Genética , Factor A de Crecimiento Endotelial Vascular/fisiología , Tumor de Wilms/genética , Tumor de Wilms/patología , Tumor de Wilms/fisiopatologíaRESUMEN
In the current study, we have cultured and propagated the cells obtained from the granulation tissue that forms around perforated polyvinyl tubes placed in the subcutaneous space of normal rats. We found that these cells (called granulation tissue-derived stem cells [GTSCs]) expressed markers of embryonic pluripotent cells (Oct-4 and Nanog) and of adult stem cells (CXCR4 and Thy1.1) as well as produced high levels of vascular endothelial growth factor (VEGF) for up to 10 passages. By fluorescence-activated cell-sorting (FACS) analysis, GTSCs were positive for stem-cell surface markers CD90, CD59, and CD44 and were negative for CD45, which suggests that they were of mesenchymal origin and not of hematopoietic lineage. When incubated in specific differentiation medium, these cells transformed into adipogenic, osteogenic, and chondrogenic lineages, which shows that they were multipotent. Furthermore, after systemic injection, these cells were found in the vicinity of an injured site created in the liver but not in normal areas of the liver, which indicates their propensity to seek and engraft to an injured area in the body. We conclude that granulation tissue induced by a large foreign body is a convenient source of adult stem cells that can be maintained in culture and can be used to repair and regenerate injured tissue.
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Células Madre Adultas/citología , Cuerpos Extraños/patología , Tejido de Granulación/citología , Polivinilos/efectos adversos , Adipocitos/citología , Células Madre Adultas/metabolismo , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Forma de la Célula , Células Cultivadas , Condrocitos/citología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Citometría de Flujo , Tejido de Granulación/metabolismo , Hígado/patología , Osteocitos/citología , Ratas , Trasplante de Células Madre , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
When rat omentum becomes activated by intraperitoneal injection of inert polydextran particles, these particles are rapidly surrounded by cells that express markers of adult stem cells (SDF-1alpha, CXCR4, WT-1) and of embryonic pluripotent cells (Oct-4, Nanog, SSEA-1). We have cultured such cells, because they may offer a convenient source of adult stem cells, and have found that they retain stem cell markers and produce high levels of vascular endothelial growth factor for up to ten passages. After systemic or local injection of these cultured cells into rats with acute injury of various organs, the cells specifically engraft at the injured sites. Thus, our experiments show that omental stromal cells can be cultured from activated omentum, and that these cells exhibit stem cell properties enabling them to be used for repair and possibly for the regeneration of damaged tissues.
Asunto(s)
Células Madre Adultas/citología , Antígenos de Diferenciación/metabolismo , Movimiento Celular , Epiplón/citología , Células del Estroma/citología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Células Madre Adultas/metabolismo , Células Madre Adultas/trasplante , Animales , Técnicas de Cultivo de Célula , Separación Celular , Células Cultivadas , Quimiocina CXCL12/metabolismo , Dextranos/administración & dosificación , Dextranos/farmacología , Tejido de Granulación/citología , Inyecciones Intraperitoneales , Riñón/patología , Antígeno Lewis X/metabolismo , Epiplón/efectos de los fármacos , Epiplón/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores CXCR4/metabolismo , Daño por Reperfusión/patología , Trasplante de Células Madre , Células del Estroma/metabolismo , Células del Estroma/trasplante , Proteínas WT1/metabolismoRESUMEN
In order to study the mechanism by which an omental pedicle promotes healing when applied to an injured site, we injected a foreign body into the abdominal cavity to activate the omentum. One week after the injection, we isolated the omentum and measured blood vessel density, blood content, growth and angiogenesis factors (VEGF and others), chemotactic factors (SDF-1 alpha), and progenitor cells (CXCR-4, WT-1). We found that the native omentum, which consisted mostly of adipose tissue, expanded the mass of its non-adipose part (milky spots) 15- to 20-fold. VEGF and other growth factors increased by two- to four-fold, blood vessel density by three-fold, and blood content by two-fold. The activated omentum also showed increases in SDF-1 alpha, CXCR-4, and WT-1 cells (factors and cells positively associated with tissue regeneration). Thus, we propose that an omentum activated by a foreign body (or by injury) greatly expands its milky-spot tissue and becomes rich in growth factors and progenitor cells that facilitate the healing and regeneration of injured tissue.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Epiplón/fisiología , Regeneración/fisiología , Cicatrización de Heridas/fisiología , Animales , Vasos Sanguíneos/crecimiento & desarrollo , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Cuerpos Extraños/metabolismo , Humanos , Epiplón/irrigación sanguínea , Epiplón/citología , Epiplón/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores CXCR4/metabolismo , Flujo Sanguíneo Regional , Células Madre/citología , Células Madre/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas WT1/metabolismoRESUMEN
To determine if pancreatic progenitor cells can be induced to form insulin producing cells in vivo, we auto-transplanted fragments of streptozotocin-induced diabetic pancreas into omentum pre-injected with a foreign material. As shown previously, omentum pre-activated in this manner becomes rich in growth factors and progenitor cells. After auto-transplanting diabetic pancreas in the activated omentum, new insulin secreting cells appeared in the omentum in niches surrounding the foreign particles--a site previously shown to harbor progenitor cells. Extracts of these omenta contained measurable insulin. Four of eight diabetic animals treated in this manner became normoglycemic. This shows that new insulin producing cells can be regenerated from diabetic pancreas by auto-transplanting pancreatic fragments into the activated omentum, an environment rich in growth factors and progenitor cells.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Insulina/metabolismo , Epiplón/fisiología , Trasplante de Páncreas/fisiología , Animales , Secreción de Insulina , Masculino , Epiplón/metabolismo , Pancreatectomía , Ratas , Ratas Sprague-Dawley , Trasplante AutólogoRESUMEN
To understand impaired angiogenesis in diabetic wounds, polyvinyl tubes were implanted subcutaneously in rats to form a granulation tissue for 2 weeks and the granulation tissue was studied after inducing diabetes with streptozotocin. By 1 week of diabetes, the granulation tissue was bloody and thinner than controls, its medial layer was depleted of microvessels, and the surviving vessels appeared dehisced. Vascular endothelial growth factor (VEGF) in the diabetic granulation tissue was reduced by 50% compared with control granulation tissue. After 3 days of diabetes, the diabetic tissue showed a greater degree of apoptosis in the microvessels. Chemotactic factors [stromal cell-derived factor-1alpha and chemokine receptor-4 (CXCR-4)], responsible for attracting bone marrow cells, showed equal intensity in control and diabetic tissues. As expected, progenitor endothelial CD-34 cells were observed in abundance in both the control and the diabetic granulation tissues. However, although the CD-34-positive cells appeared mostly to be integrated in the blood vessels of the control tissue, fewer such cells were present in the blood vessels of the diabetic tissues, suggesting that CD-34 failed to integrate into new blood vessels. Infusion of VEGF in the granulation tissue of diabetic rats for 1 week resulted in complete prevention of the microvascular defect compared with the contralateral granulation tissue that showed the typical diabetic changes. It was concluded that diabetes causes reduction of VEGF in the wound, resulting in loss of blood vessels by apoptosis and possible failure of CD-34 cells to integrate into the vessel structure.