RESUMEN
BACKGROUND: Using a proteomic approach, we aimed to identify and compare the urinary excretion of proteins involved in lipid transport and metabolism in children with kidney stones and hypercalciuria (CAL), hypocitraturia (CIT), and normal metabolic work-up (NM), and in healthy controls (HCs). Additionally, we aimed to confirm these results using ELISA, and to examine the relationship between the urinary excretion of selected proteins with demographic, dietary, blood, and urinary parameters. METHODS: Prospective, controlled, pilot study of pooled urine from CAL, CIT, and NM versus age- and gender-matched HCs, using liquid chromatography-mass spectrometry. Relative protein abundance was estimated using spectral counting. Results were confirmed by ELISA performed on individual samples. RESULTS: Of the 1,813 proteins identified, 230 met the above criteria. Of those, 5 proteins (apolipoprotein A-II [APOA2]; apolipoprotein A-IV [APOA4]; apolipoprotein C-III [APOA3]; fatty acid-binding protein, liver [FABPL]; fatty acid-binding protein, adipocyte [FABP4]) involved in lipid metabolism and transport were found in the CAL group, with significant differences compared with HCs. ELISA analysis indicated statistically significant differences in the urinary excretion of APOC3, APOA4, and FABPL in the CAL group compared with HCs. Twenty-four-hour urinary calcium excretion correlated significantly with concentrations of ApoC3 (r = 0.77, p < 0.001), and FABPL (r = 0.80, p = 0.005). CONCLUSIONS: We provide proteomic data showing increased urinary excretion of lipid metabolism/transport-related proteins in children with kidney stones and hypercalciuria. These findings suggest that abnormalities in lipid metabolism might play a role in kidney stone formation.
Asunto(s)
Apolipoproteínas/orina , Hipercalciuria/orina , Cálculos Renales/orina , Eliminación Renal , Adolescente , Apolipoproteínas/metabolismo , Calcio/metabolismo , Calcio/orina , Niño , Cromatografía Liquida , Citratos/orina , Ensayo de Inmunoadsorción Enzimática , Proteínas de Unión a Ácidos Grasos/metabolismo , Femenino , Humanos , Hipercalciuria/metabolismo , Cálculos Renales/metabolismo , Masculino , Espectrometría de Masas , Proyectos Piloto , Estudios Prospectivos , Proteómica/métodosRESUMEN
AIM: To investigate whether apoM is excreted in urine of children undergoing heart surgery and the potential of apoM as early biomarker of acute kidney injury (AKI). MATERIALS & METHODS: Urine was collected in children undergoing heart surgery. ApoM was measured with ELISA. U-apoM was characterized by gel filtration chromatography and western blotting. RESULTS: ApoM was excreted into the urine 0-4 h postoperatively as the full-length apoM in particles smaller than plasma HDL. At 0 h, U-apoM predicted AKI with an area under the receiver-operating characteristics curve of 0.70 (p < 0.018). Sensitivity was 0.71 and specificity was 0.68 at a cutoff level at 1.45 nmol/l. CONCLUSION: ApoM is excreted in the urine of children after cardiac surgery. Its potential as biomarker of AKI deserves exploration.
Asunto(s)
Lesión Renal Aguda/cirugía , Lesión Renal Aguda/orina , Apolipoproteínas/orina , Procedimientos Quirúrgicos Cardíacos , Lipocalinas/orina , Proteínas de Fase Aguda/orina , Apolipoproteínas M , Biomarcadores/orina , Niño , Preescolar , Estudios de Cohortes , Femenino , Humanos , Lactante , Recién Nacido , Lipocalina 2 , Masculino , Proteínas Proto-Oncogénicas/orinaRESUMEN
BACKGROUND: It has been well documented that apolipoprotein M (apoM) is principally expressed in hepatocytes as well as renal tubular epithelial cells. The importance of apoM in the kidney is unknown. In the present study we examined urinary any apoM after short-term ischemia-reperfusion injury (IRI) of kidney in a rat model. METHODS: The kidneys of 11 male Sprague-Dawley rats were rendered ischemic for 45 minutes followed by different intervals of reperfusion. Serum and urine apoM concentrations were determined using a dot-blot analysis with specific rabbit anti-human apoM antibodies that cross-react with rat apoM. Serum concentrations of blood urea nitrogen (BUN) and creatinine (Cr) were determined using standard clinical automated analyses. RESULTS: BUN was significantly elevated after 45 minutes of ischemia followed by 24 hours of reperfusion; serum Cr concentrations were also significantly increased at 6 and 24 hours of reperfusion. Interestingly, similar to BUN and Cr, serum apoM concentrations were significantly increased after ischemia for 45 minutes alone and after 2 hours of reperfusion. Urinary apoM concentrations were obviously increased after 2 h as well as 6 hours of reperfusion. CONCLUSION: apoM showed characteristics of an acute-phase reactive protein; its occurrence in urine may be considered to be a biomarker of acute renal injury.
Asunto(s)
Lesión Renal Aguda/orina , Proteínas de Fase Aguda/orina , Apolipoproteínas/orina , Lipocalinas/orina , Daño por Reperfusión/orina , Lesión Renal Aguda/sangre , Lesión Renal Aguda/diagnóstico , Animales , Apolipoproteínas/sangre , Apolipoproteínas M , Biomarcadores/sangre , Biomarcadores/orina , Nitrógeno de la Urea Sanguínea , Creatinina/sangre , Modelos Animales de Enfermedad , Lipocalinas/sangre , Masculino , Valor Predictivo de las Pruebas , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/sangre , Daño por Reperfusión/diagnóstico , Factores de TiempoRESUMEN
In this study, we evaluated the reproducibility of abundant urine protein depletion by hexapeptide-based library beads and an antibody-based affinity column using the iTRAQ technique. The antibody-based affinity-depletion approach, which proved superior, was then applied in conjunction with iTRAQ to discover proteins that were differentially expressed between pooled urine samples from hernia and bladder cancer patients. Several proteins, including seven apolipoproteins, TIM, SAA4, and proEGF were further verified in 111 to 203 individual urine samples from patients with hernia, bladder cancer, or kidney cancer. Six apolipoproteins (APOA1, APOA2, APOB, APOC2, APOC3, and APOE) were able to differentiate bladder cancer from hernia. SAA4 was significantly increased in bladder cancer subgroups, whereas ProEGF was significantly decreased in bladder cancer subgroups. Additionally, the combination of SAA4 and ProEGF exhibited higher diagnostic capacity (AUC=0.80 and p<0.001) in discriminating bladder cancer from hernia than either marker alone. Using MetaCore software to interpret global changes of the urine proteome caused by bladder cancer, we found that the most notable alterations were in immune-response/alternative complement and blood-coagulation pathways. This study confirmed the clinical significance of the urine proteome in the development of non-invasive biomarkers for the detection of bladder cancer. BIOLOGICAL SIGNIFICANCE: In this study, we evaluated the reproducibility of abundant urine protein depletion by hexapeptide-based library beads and an antibody-based affinity column using the iTRAQ technique. The antibody-based affinity-depletion approach, which proved superior, was then applied in conjunction with iTRAQ to discover proteins that were differentially expressed between pooled urine samples from hernia and bladder cancer patients. Several proteins, including seven apolipoproteins, TIM, SAA4, and proEGF were further verified in 111 to 203 individual urine samples from patients with hernia, bladder cancer, or kidney cancer. SAA4 was significantly increased in bladder cancer subgroups, whereas ProEGF was significantly decreased in bladder cancer subgroups. Additionally, the combination of SAA4 and ProEGF exhibited higher diagnostic capacity in discriminating bladder cancer from hernia than either marker alone. A marker panel composed by two novel biomarker candidates, SAA4 and proEGF, was first discovered and verified successfully using Western blotting. To the best of our knowledge, the associations of urinary SAA4 and proEGF with bladder tumor and kidney cancer have not been mentioned before. In the present study, we discovered and verified SAA4 and proEGF as potential bladder cancer biomarker for the first time.
Asunto(s)
Apolipoproteínas/orina , Biomarcadores de Tumor/orina , Factor de Crecimiento Epidérmico/orina , Proteínas de Neoplasias/orina , Proteína Amiloide A Sérica/orina , Neoplasias de la Vejiga Urinaria/orina , Femenino , Humanos , Masculino , Proteoma/metabolismo , Proteómica/métodos , Programas InformáticosRESUMEN
Apolipoprotein (apo) M is a novel apolipoprotein belonging to the lipocalin protein superfamily, i.e. proteins binding small lipophilic compounds. Like other apolipoproteins, it is expressed in hepatocytes and secreted into plasma where it associates with high-density lipoprotein particles. In addition, apoM is expressed at high levels in the kidney tubule cells. In this study, we show that the multiligand receptor megalin, which is expressed in kidney proximal tubule cells, is a receptor for apoM and mediates its uptake in the kidney. To examine apoM binding to megalin, a recombinant apoM was expressed in Escherichia coli and used in surface plasmon resonance and cell culture studies. The results showed apoM binding to immobilized megalin [dissociation constant (Kd) approximately 0.3-1 microm] and that the apoM was endocytosed by cultured rat yolk sac cells in a megalin-dependent manner. To examine the importance of apoM binding by megalin in vivo, we analyzed mice with a tissue-specific deficiency of megalin in the kidney. Megalin deficiency was associated with pronounced urinary excretion of apoM, whereas apoM was not detected in normal mouse, human, or rat urine. Gel filtration analysis showed that the urinary apoM-containing particles were small and devoid of apoA-I. The results suggest that apoM binds to megalin and that megalin-mediated endocytosis in kidney proximal tubules prevents apoM excretion in the urine.
Asunto(s)
Apolipoproteínas/metabolismo , Riñón/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Animales , Apolipoproteína A-I/metabolismo , Apolipoproteína A-I/orina , Apolipoproteínas/sangre , Apolipoproteínas/orina , Apolipoproteínas M , Células Cultivadas , Endocitosis , Humanos , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/deficiencia , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Ratones , Ratones Transgénicos , Unión Proteica , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de Superficie , Saco Vitelino/citologíaRESUMEN
BACKGROUND: Plasma lipoprotein (a) [Lp(a)] has been shown to be a risk factor for atherosclerosis in numerous studies. However, the catabolism of this lipoprotein is not very clear. We and others have shown that Lp(a) is excreted into urine in the form of fragments. Lp(a) has also been shown to exist in a low-density non-lipoprotein (LDL)-bound form. Since Lp(a) is increased in all forms of kidney disease with reduced excretory kidney function and decreased excretion of apo(a) fragments could be partially responsible for this increase, we investigated the relationship of non-LDL-bound apo(a), urinary apo(a) fragments and plasma Lp(a) in patients with impaired renal function. METHODS: Plasma Lp(a), non-LDL-bound apo(a) and urinary apo(a) fragments were measured in 55 kidney disease patients (28 males and 27 females) and matched controls. RESULTS: Plasma Lp(a) and non-LDL-bound apo(a) were increased in patients, whereas urinary apo(a) was decreased, especially in patients with a creatinine clearance < 70 ml/min. There was a significant correlation between plasma Lp(a) and non-LDL-bound apo(a) in patients and controls. CONCLUSION: We conclude that decreased urinary apo(a) excretion could be one possible mechanism of increased plasma Lp(a) and non-LDL-bound apo(a) in patients with decreased kidney function.
Asunto(s)
Apolipoproteínas/sangre , Apolipoproteínas/orina , Enfermedades Renales/sangre , Enfermedades Renales/orina , Lipoproteína(a)/sangre , Lipoproteína(a)/orina , Adulto , Anciano , Apoproteína(a) , Estudios de Casos y Controles , Creatinina/sangre , Femenino , Humanos , Enfermedades Renales/fisiopatología , Masculino , Persona de Mediana EdadRESUMEN
The atherogenic lipoprotein Lp(a) consists of an LDL-like core and apo(a), linked to apoB via a thiol bridge. Apo(a) fragments ranging in size from 60 to 220 kDa are excreted into urine and the excretion rate correlates significantly with the plasma levels of Lp(a). In order to study the interrelationship of apo(a) secretion with that of other plasma proteins, urinary apo(a) and protein secretion of five probands were followed for 24 h at different urinary densities. The excretion rate of apo(a) fragments, despite their high molecular weight, was highest, followed by apoD, orosomucoid, albumin and beta(2)-glycoprotein-I (beta2-GI) and plasminogen (1.58, 0.87, 0.095, 0.027, 0.013 and <0.001%/day, respectively). There was a highly significant correlation between apo(a), apoD and beta2-GI concentrations but not with albumin and orosomucoid concentrations in urine. The only protein that was fragmented in urine was apo(a) while the other proteins had molecular weights comparable to those in plasma. We conclude that a previously suggested fragmentation of apo(a) by the kidney is not a rate-limiting step in its excretion. Since plasminogen, another kringle-IV-containing plasma compound, and fragments thereof, are undetectable in urine under identical experimental conditions, it is very unlikely that the characteristic kringle structure is responsible for the high excretion rate of apo(a).
Asunto(s)
Apolipoproteínas/orina , Proteínas Sanguíneas/orina , Lipoproteína(a)/orina , Adulto , Apoproteína(a) , Western Blotting , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estándares de Referencia , Sensibilidad y EspecificidadRESUMEN
Fifty patients with stable slight and moderate uncomplicated essential hypertension, treated by ramipril, atenolol, or isradipine, were examined. Total protein and urinary excretion of individual proteins were studied before and after treatment. Urinary concentrations of apolipoproteins A1 and B1, alpha 1-acid glycoprotein, alpha 1-antitrypsin, prealbumin, albumin, beta 2-microglobulin, transferrin, haptoglobin, IgG and IgA, and C3 and C4 complement components were measured. Index of proteinuria selectiveness was calculated for each portion of urine. All three drugs exerted a nephroprotective effect, atenolol being the most active of them. Apolipoproteins, IgG, and complement components were the most valuable for diagnosis. Their excretion correlated with the severity of arterial hypertension and efficiency of treatment. Use of protein markers helps reliably assess the renal function and monitor the treatment efficiency.
Asunto(s)
Antihipertensivos/farmacología , Apolipoproteínas/orina , Complemento C3/orina , Complemento C4/orina , Hipertensión/tratamiento farmacológico , Inmunoglobulina G/orina , Riñón/efectos de los fármacos , Proteinuria/diagnóstico , Adulto , Anciano , Albuminuria/diagnóstico , Antihipertensivos/uso terapéutico , Atenolol/farmacología , Atenolol/uso terapéutico , Biomarcadores , Femenino , Haptoglobinas/orina , Humanos , Inmunoglobulinas/orina , Isradipino/farmacología , Isradipino/uso terapéutico , Masculino , Persona de Mediana Edad , Modelos Teóricos , Monitoreo Fisiológico , Prealbúmina/orina , Ramipril/farmacología , Ramipril/uso terapéutico , Transferrina/orinaRESUMEN
Plasma levels of lipoprotein(a) (Lp(a)), an atherogenic particle, are elevated in kidney disease, which suggests a role of this organ in the metabolism of Lp(a). Additional evidence for a role of the kidney in the clearance of Lp(a) is provided by the fact that circulating N-terminal fragments of apolipoprotein(a) (apo(a)) are processed and eliminated by the renal route. To further understand the mechanism underlying such renal excretion, the levels of apo(a) fragments in plasma and urine relative to plasma Lp(a) levels were determined in patients with nephrotic syndrome (n = 15). In plasma, the absolute (24.7 +/- 20.4 versus 2.16 +/- 2.99 microg/ml, P < 0.0001) as well as the relative amounts of apo(a) fragments (4.6 +/-3.4% versus 2.1 +/- 3.3% of total Lp(a), P < 0.0001) were significantly elevated in nephrotic patients compared with a control, normolipidemic population. In addition, urinary apo(a) excretion in patients with nephrotic syndrome was markedly elevated compared with that in control subjects (578 +/- 622 versus 27.7 +/- 44 ng/ml per mg creatinine, P < 0.001). However, the fractional catabolic rates of apo(a) fragments were similar in both groups (0.68 +/- 0.67% and 0.62 +/- 0.47% in nephrotic and control subjects, respectively), suggesting that increased plasma concentrations of apo(a) fragments in nephrotic subjects are more dependent on the rate of synthesis rather than on the catabolic rate. Molecular analysis of apo(a) immunoreactive material in urine revealed that the patterns of apo(a) fragments in nephrotic patients were distinct from those of control subjects. Full-length apo(a), large N-terminal apo(a) fragments similar in size to those present in plasma, as well as C-terminal fragments of apo(a) were detected in urine from nephrotic patients but not in urine from controls. All of these apo(a) forms were in addition to smaller N-terminal apo(a) fragments present in normal urine. This study also demonstrated the presence of Lp(a) in urine from nephrotic patients by ultracentrifugal fractionation. These data suggest that in nephrotic syndrome, Lp(a) and large fragments of apo(a) are passively filtered by the kidney through the glomerulus, whereas smaller apo(a) fragments are secreted into the urine.
Asunto(s)
Lipoproteína(a)/sangre , Lipoproteína(a)/orina , Síndrome Nefrótico/sangre , Síndrome Nefrótico/orina , Adulto , Apolipoproteínas/sangre , Apolipoproteínas/orina , Apoproteína(a) , Electroforesis en Gel Bidimensional , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Immunoblotting , Lípidos/sangre , Masculino , Persona de Mediana Edad , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/orina , UltracentrifugaciónRESUMEN
Increased plasma lipoprotein (a) (Lp(a)) levels are strongly associated with premature cardiovascular disease and stroke. Recently we, as well as other groups, found that apolipoprotein (a) (apo(a)) fragments appear in the urine of healthy individuals, and that renal transplant patients with impaired renal function excrete fewer apo(a) fragments into their urine compared with controls. As the excretion mode of apo(a) is presently unknown, we determined plasma Lp(a) levels and urinary apo(a) excretion in relation to kidney function in 58 proteinuric patients and 58 healthy controls. For the first time, urinary apo(a) excretion was related to apo(a) isoforms. Plasma Lp(a) values were higher in the proteinuric patients compared with the controls, independent of their renal function. The patients with low-molecular-weight apo(a) isoforms had higher Lp(a) plasma levels, whereas the patients with high-molecular-weight apo(a) isoforms had lower Lp(a) plasma levels. Urinary apo(a) showed a very similar pattern to that of plasma Lp(a), being significantly higher in patients with low-molecular-weight isoforms as compared with patients with high-molecular-weight isoforms. Urinary apo(a) excretion was significantly decreased in the patient group when compared with healthy controls. There was a close correlation (P < 0.001) between the plasma Lp(a) and urinary apo(a) excretion in both the patient group and the control group. Urinary apo(a) excretion did not correlate with protein excretion, creatinine clearance or plasma creatinine levels. We conclude that urinary apo(a) excretion correlates with plasma Lp(a) and Lp(a) isoforms, and that proteinuric patients excrete significantly less apo(a) into their urine than healthy controls, a factor that might contribute to increased plasma Lp(a) levels in these patients.
Asunto(s)
Apolipoproteínas/sangre , Apolipoproteínas/orina , Lipoproteínas/sangre , Lipoproteínas/orina , Proteinuria/metabolismo , Adulto , Anciano , Análisis de Varianza , Femenino , Humanos , Fallo Renal Crónico/sangre , Fallo Renal Crónico/orina , Pruebas de Función Renal , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , Estadísticas no ParamétricasRESUMEN
Apolipoprotein(a) [apo(a)] contains multiple kringle 4 repeats and circulates as part of lipoprotein(a) [Lp(a)]. Apo(a) is synthesized by the liver but its clearance mechanism is unknown. Previously, we showed that kringle 4-containing fragments of apo(a) are present in human urine. To probe their origin, human plasma was examined and a series of apo(a) immunoreactive peptides larger in size than urinary fragments was identified. The concentration of apo(a) fragments in plasma was directly related to the plasma level of Lp(a) and the 24-h urinary excretion of apo(a). Individuals with low (< 2 mg/dl) plasma levels of Lp(a) had proportionally more apo(a) circulating as fragments in their plasma. Similar apo(a) fragments were identified in baboon plasma but not in conditioned media from primary cultures of baboon hepatocytes, suggesting that the apo(a) fragments are generated from circulating apo(a) or Lp(a). When apo(a) fragments purified from human plasma were injected intravenously into mice, a species that does not produce apo(a), apo(a) fragments similar to those found in human urine were readily detected in mouse urine. Thus, we propose that apo(a) fragments in human plasma are derived from circulating apo(a)/Lp(a) and are the source of urinary apo(a).
Asunto(s)
Apolipoproteínas/metabolismo , Kringles/inmunología , Animales , Apolipoproteínas/sangre , Apolipoproteínas/química , Apolipoproteínas/orina , Células Cultivadas , Medios de Cultivo Condicionados/análisis , Medios de Cultivo Condicionados/química , Heparina/metabolismo , Humanos , Immunoblotting , Isomerismo , Riñón/fisiología , Hígado/citología , Ratones , Ratones Transgénicos , PapioRESUMEN
A zone immunoelectrophoresis assay (ZIA) has been developed for the quantification of apolipoprotein D (apo D) in human unconcentrated cerebrospinal fluid (CSF). The apo D concentrations of samples of the serum, plasma and CSF were directly proportional to the migration distances of the corresponding zones of immunoprecipitates developed during electrophoresis in glass capillaries filled with antibody-containing agarose gel. A linear standard curve, between about 1 and 12 mg of apo D/1 was obtained using a commercial serum preparation. Seronorm, as apo D standard. The coefficients of variation of the ZIA were below 8% (n = 5 x 6) and 10% (n = 8) for within-run and between-run reproducibility, respectively. Quantification experiments with disulfide-reducing agent, mixtures of CSF and urine as well as frozen and stored CSF samples indicated parallelism between the precipitate-forming immunologic reactions of apo D in different sample matrices when performed with ZIA. Application of this method to quantify apo D of CSF and plasma samples from 51 normal healthy men aged 16-72 years yielded means +/- SD of 5.3 +/- 1.5 mg/l and 128.4 +/- 22.7 mg/l, respectively. No correlation was found between the CSF and plasma apo D concentrations.
Asunto(s)
Apolipoproteínas/líquido cefalorraquídeo , Adolescente , Adulto , Anciano , Apolipoproteínas/orina , Apolipoproteínas D , Humanos , Inmunoelectroforesis/métodos , Masculino , Persona de Mediana Edad , Estándares de ReferenciaRESUMEN
Apo(a) is a large glycoprotein of unknown function that circulates in plasma as part of lipoprotein(a). Apo(a) is structurally related to plasminogen and contains at least 10 kringle (K)4 repeats (type 1-10), a K5 repeat and sequences similar to the protease domain of plasminogen. Plasminogen generates two biologically active peptides: plasmin and angiostatin, a kringle-containing peptide. As a first step in determining if apo(a) generates a similar kringle-containing peptide, human urine was immunologically examined. Fragments ranging in size from 85 to 215 kD were immunodetected using antibodies directed against epitopes in the K4-type 2 repeat, but not the K4-type 9 repeat or protease domain, NH2-terminal sequence analysis revealed sequences specific for the K4-type 1 repeat, confirming that the fragments are from the NH2 terminus of the K4 array. The amount of urinary apo(a) rose in proportion to the plasma lipoprotein(a) concentration. Even individuals with trace to no apo(a) in plasma had immunodetectable apo(a) fragments in their urine. Intravenous administration of the human urinary apo(a) into mice resulted in the urine. These findings suggest that the apo(a) fragments found in urine are formed extrarenally and then excreted by the kidney.
Asunto(s)
Apolipoproteínas/orina , Kringles , Lipoproteína(a) , Fragmentos de Péptidos/orina , Secuencia de Aminoácidos , Animales , Apolipoproteínas/sangre , Apolipoproteínas/química , Apolipoproteínas/inmunología , Apoproteína(a) , Humanos , Immunoblotting , Riñón/metabolismo , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Análisis de SecuenciaRESUMEN
Apolipoprotein (apo) D is a glycoprotein that contains at least one free cysteine. This allows the formation of disulfide linked dimers of apoD, a phenomenon that could interfere with the study of the isoforms of apoD. Consequently, it is important to consider the effects of hetero- and homodimer formation on the molecular heterogeneity of apoD as well as on the evaluation of the specificity of antibodies to this glycoprotein. The identification of apoD in urine has provided a potential new marker of tubular proteinuria. Thus, we have studied the specificity of our polyclonal antibodies to apoD against the proteins present in normal urine, and at the same time, the existence of dimeric species of apoD linked by disulfide bonds in urine. The specimens were obtained from apparently healthy individuals and analyzed by Western blot. The results showed that apoD in urine exists as a mixture of monomers and dimers, the latter having apparent molecular weights different from those occurring in plasma. Only monomeric apoD was observed under reducing conditions, proving the monospecificity of the polyclonal apoD antibodies.
Asunto(s)
Apolipoproteínas/orina , Disulfuros/química , Adulto , Apolipoproteínas/química , Apolipoproteínas D , Humanos , Masculino , Conformación ProteicaRESUMEN
AIM: To compare the urinary excretion of beta 2-glycoprotein-1 with that of two other markers of early tubular disorder in diabetic patients without clinical proteinuria. METHODS: The urinary excretion of retinol binding protein, beta 2-glycoprotein-1, and N-acetyl-beta-D-glucosaminidase was measured in 90 known diabetic patients who had a negative reagent strip test for proteinuria. RESULTS: Among 43 patients with urinary albumin excretion within the reference range, 23 (53%) had raised urinary N-acetyl-beta-D-glucosaminidase activity, five (12%) increased excretion of beta 2-glycoprotein-1, and five (12%) increased loss of retinol binding protein. Among 47 patients with an albumin excretion of 0.9-7.9 mg/mmol creatinine, 42 (89%) had increased urinary N-acetyl-beta-D-glucosaminidase, 23 (49%) an increased output of beta 2-glycoprotein-1, and 16 (34%) a raised excretion of retinol binding protein. The excretion of these markers of tubular defects seldom exceeded two and a half times the upper reference limit and the differences between the findings in the insulin dependent and non-insulin dependent patients with similar albumin excretion were small and insignificant. CONCLUSIONS: In diabetic patients with a negative dipstick test for proteinuria: (a) assay of urinary beta 2-glycoprotein-1 may be a more sensitive test for the detection of impaired tubular reabsorption of protein than measurement of retinol-binding protein; (b) assay of N-acetyl-beta-D-glucosaminidase can detect tubular injury at a time when protein reabsorption remains normal; and (c) impaired renal tubular function may be present in the absence of evidence of glomerular malfunction.
Asunto(s)
Apolipoproteínas/orina , Diabetes Mellitus/orina , Glicoproteínas/orina , Túbulos Renales/fisiopatología , Acetilglucosaminidasa/orina , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/orina , Diabetes Mellitus/fisiopatología , Diabetes Mellitus Tipo 1/orina , Diabetes Mellitus Tipo 2/orina , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Unión al Retinol/orina , beta 2 Glicoproteína IRESUMEN
Apolipoprotein D is a previously unrecognized urinary protein of unknown function which we have tested as a potential marker for kidney malfunction. This protein and alpha 1-microglobulin have been quantified by zone immunoelectrophoresis assay in urine samples from a group of eight men occupationally exposed to cadmium-containing welding fumes for many years. All these workers had highly elevated concentrations of urinary cadmium and indications of tubular proteinuria, as compared to a group of 50 apparently healthy normal men analyzed in parallel. The cadmium-exposed workers demonstrated three- and 15-fold average increases in apolipoprotein D and alpha 1-microglobulin, respectively, over normal values in urine, estimated both as excretion rates and as milligrams of protein per mmol of creatinine. All these increments were highly significantly different (P < 0.001) from the corresponding values of the reference group. Essentially the same results were obtained for each of the proteins from two independent consecutive samplings of the workers' urine. There were good linear (R = 0.70, 0.80) and logarithmic (R = 0.84, 0.81) correlations between the urinary concentrations of alpha 1-microglobulin and apolipoprotein D for both the reference and the study group. Although not as sensitive an indicator for tubular proteinuria as alpha 1-microglobulin, apolipoprotein D, being a storage-stable urinary protein, seems a valuable complement for the diagnosis of tubular malfunction.
Asunto(s)
alfa-Globulinas/orina , Apolipoproteínas/orina , Cadmio/efectos adversos , Exposición Profesional/efectos adversos , Inhibidores de Proteasas/orina , Soldadura , Adulto , Apolipoproteínas D , Biomarcadores , Cadmio/sangre , Cadmio/orina , Estudios de Casos y Controles , Humanos , Masculino , Persona de Mediana Edad , Enfermedades Profesionales/inducido químicamente , Enfermedades Profesionales/orina , Proteinuria/inducido químicamente , Proteinuria/orinaRESUMEN
AIM: To determine whether urinary beta 2-glycoprotein-1 assays can provide improved discrimination between chronic renal diseases which are primarily of tubular or glomerular origin. METHODS: Urinary beta 2-glycoprotein-1, retinol-binding protein, alpha 1-microglobulin, beta 2-microglobulin, N-acetyl-beta-D-glucosa-minidase and albumin were measured in 51 patients with primary glomerular disease, 23 with obstructive nephropathy, and 15 with polycystic kidney disease, and expressed per mmol of creatinine. Plasma beta 2-glycoprotein-1 was assayed in 52 patients and plasma creatinine in all 89. The findings were compared between the diagnostic groups and with previously published data relating to primary tubular disorders. RESULTS: All 31 patients with plasma creatinine greater than 200 mumol/l excreted increased amounts of beta 2-glycoprotein-1, retinol-binding protein, and alpha 1-microglobulin, and 29 had increased N-acetyl-beta-D-glucosaminidase; the quantities were generally similar to those found in comparable patients with primary tubular pathology. Among 58 with plasma creatinine concentrations under 200 mumol/l, increases in beta 2-glycoprotein-1, retinol-binding protein, and alpha 1-microglobulin excretion were less common and much smaller, especially in those with obstructive nephropathy and polycystic disease. The ratios of the excretion of albumin to the other proteins provided the clearest discrimination between the patients with glomerular or tubular malfunction, but an area of overlap was present which embraced those with obstructive nephropathy and polycystic disease. CONCLUSIONS: Increased excretion of beta 2-glycoprotein-1 due to a raised plasma concentration or diminution of tubular reabsorption, or both, is common in all the forms of renal disease investigated, and both plasma creatinine and urinary albumin must be taken into account when interpreting results. Ratios of urinary albumin: beta 2-glycoprotein-1 greater than 1000 are highly suggestive of primary glomerular disease and those less than 40 of primary tubular disease. Used in this way, beta 2-glycoprotein-1 assays provide superior discrimination between glomerular and tubular malfunction when compared with retinol binding protein but the best discrimination is provided by albumin: alpha 1-microglobulin ratios.
Asunto(s)
Apolipoproteínas/orina , Glicoproteínas/orina , Enfermedades Renales/orina , Acetilglucosaminidasa/orina , Adolescente , Adulto , Anciano , Albuminuria , alfa-Globulinas/orina , Enfermedad Crónica , Creatinina/sangre , Femenino , Humanos , Enfermedades Renales/sangre , Enfermedades Renales/fisiopatología , Glomérulos Renales/fisiopatología , Túbulos Renales/fisiopatología , Masculino , Persona de Mediana Edad , Inhibidores de Proteasas/orina , Proteínas de Unión al Retinol/orina , Proteínas Plasmáticas de Unión al Retinol , beta 2 Glicoproteína I , Microglobulina beta-2/orinaRESUMEN
A zone immunoelectrophoresis assay (ZIA) has been developed for the quantification of apolipoprotein D (apo D) in unconcentrated native human urine. A standard curve, linear between 1 and 8 mg apo D/l was obtained with ZIA. The relative coefficients of variation for this method were 5-9% (n = 15 x 6) with a mean +/- SD of 7 +/- 1.4% and below 11% (n = 6 x 15) for within-run and between-run reproducibility, respectively. Equal amounts of apo D in unconcentrated and diluted urines, in serum and of the purified protein produced the same zone migration distances indicating parallelism between the immunologic reactions of apo D in different sample matrixes. Storage experiments with normal urines demonstrated good stability of apo D in both acidic and alkalinized urine over at least 2 days at +5 degrees C and during several days at -20 degrees C to -40 degrees C. Using ZIA, urine samples from 50 normal healthy men aged 23-65 years were analyzed for apo D. Mean and SD were: 2.8 +/- 2.1 mg/l, 2.6 +/- 1.8 micrograms/min and 0.24 +/- 0.13 mg/mmol for concentration, rate of excretion and mass/creatinine concentration, respectively.
Asunto(s)
Apolipoproteínas/orina , Inmunoelectroforesis , Adulto , Anciano , Apolipoproteínas/sangre , Apolipoproteínas D , Femenino , Humanos , Inmunoelectroforesis/métodos , Cinética , Masculino , Persona de Mediana Edad , Pruebas de PrecipitinaRESUMEN
Lipoprotein excretion was investigated in the urine of hyperlipidemic rats with nephrotic syndrome induced by aminonucleoside of puromycin. Incubation with phosphatidylcholine liposomes was employed to float apoproteins not bound to lipids, by ultracentrifugation at d = 1.21 g/ml. On ultracentrifugation of whole, untreated urine, the amount of protein floated was 6-fold greater in nephrotic vs. control rats and consisted mainly of HDL-like particles. Sodium dodecylsulfate-polyacrylamide gel electrophoresis showed that control urine contained apoproteins A-I, A-II, E and traces of C, whereas in the nephrotic urine apo-E and a large amount of apo-C was found. Addition of liposomes to the ultracentrifugal d = 1.21 g/ml infranate and reflotation at the same density resulted only in slight increment in the floated apoproteins, mainly C and A-I. Addition of liposomes to the whole urine and centrifugation at d = 1.21 g/ml also did not produce a greater yield in the floated apoproteins of control or nephrotic urine. These results indicated that the urine is virtually devoid of lipid-free apoproteins and those floated from both the nephrotic and control urine are complexed with lipids. The plasma VLDL + LDL fraction of nephrotic rats, though increased in quantity, did not differ markedly in composition from that of control rats. The HDL, approximately 3-fold elevated in nephrotic rats, were poorer in esterified cholesterol and richer in phospholipids. Relative to plasma HDL, the nephrotic urine HDL were protein-rich and phospholipid-poor and appeared to be larger in particle size as suggested by the lower estimated specific volume. The modified plasma HDL in nephrosis may have a pathophysiological implication.
Asunto(s)
Apolipoproteínas/orina , Lipoproteínas HDL/metabolismo , Síndrome Nefrótico/orina , Animales , Apolipoproteínas/aislamiento & purificación , Colesterol/sangre , Hiperlipidemias/sangre , Hiperlipidemias/orina , Liposomas , Masculino , Síndrome Nefrótico/sangre , Fosfatidilcolinas , Proteinuria/sangre , Proteinuria/orina , Ratas , Triglicéridos/sangre , Orina/químicaRESUMEN
Apolipoprotein D has been identified in normal human urine, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting with monospecific antibodies. Urinary apolipoprotein D appeared as a main 33,000 u protein together with a minor fraction corresponding to its partially deglycosylated species of lower molecular mass. No high molecular mass forms of apolipoprotein D naturally occurring in plasma could be detected. The apolipoprotein D mean +/- SD concentration assayed with rocket immunoelectrophoresis, in urine samples from nine apparently healthy normal men, was 1.4 +/- 1.0 mg/L (range: 0.2-3.0 mg/L). Among the plasma apolipoproteins, apolipoprotein D behaves uniquely as regards its excretion in urine; the other apolipoproteins belonging to the A, B, D and E groups, although of low molecular masses, are present, at most, in trace amounts in normal urine.