RESUMO
BACKGROUND: As circulating plasma ATP concentrations are increased in pre-eclampsia, we tested whether increased plasma ATP is able to induce albuminuria during pregnancy. METHODS: Pregnant (day 14) and non-pregnant rats were infused with ATP (3000 microg/kg bw) via a permanent jugular vein cannula. Albuminuria was determined, and blood samples were taken for leukocyte counts, plasma ATP and plasma haemopexin activity. At Day 20 of pregnancy, rats were sacrificed, fetuses and placentas weighed and kidney and placental tissue were snap frozen for immunohistology. RESULTS: ATP infusion induced albuminuria exclusively in pregnant rats, together with increased neutrophil counts, decreased staining for glomerular sialoglycoproteins and CD39 expression, significant intraglomerular monocyte infiltration and increased glomerular intracellular adhesion molecule-1 (ICAM-1) expression. Plasma haemopexin activity was increased in saline-infused pregnant rats as compared to non-pregnant rats but was inhibited in pregnant ATP-infused rats (to non-pregnant levels). At the end of pregnancy (Day 20), increased plasma ATP level was exclusively seen in ATP-infused pregnant rats. In pregnant rats as compared with non-pregnant rats, we found decreased expression of glomerular AT-1 receptors, which was increased after ATP infusion exclusively in pregnant animals. CONCLUSION: The present study shows that ATP infusion induced a pro-inflammatory response leading to glomerular albuminuria exclusively in the pregnant rat. Why extracellular ATP showed this pro-inflammatory response exclusively in the pregnant condition is unclear but is probably related with relatively enhanced non-specific immunity and inflammatory reactions characteristic for the pregnant condition.
Assuntos
Trifosfato de Adenosina/efeitos adversos , Albuminúria/induzido quimicamente , Complicações na Gravidez/induzido quimicamente , Prenhez/metabolismo , Trifosfato de Adenosina/sangue , Albuminúria/metabolismo , Albuminúria/patologia , Animais , Antígenos CD/metabolismo , Apirase/metabolismo , Modelos Animais de Doenças , Feminino , Hemopexina/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Monócitos/patologia , Neutrófilos/patologia , Placenta/metabolismo , Gravidez , Complicações na Gravidez/metabolismo , Complicações na Gravidez/patologia , Ratos , Ratos Wistar , Receptor Tipo 1 de Angiotensina/metabolismoRESUMO
Chronic low-grade inflammation is involved in late renal transplant dysfunction. Recent studies suggest a role for hemopexin, an acute phase protein, in kidney damage. We investigated whether hemopexin activity (Hx) predicts graft failure in renal transplant recipients (RTRs). In 557 RTRs with functioning grafts for >or=1 year, Hx was measured in citrate-plasma. RTRs were divided according to Hx into two groups; A: sextile 1-5 (464 RTRs, 83%) and B: sextile 6 (92 RTRs, 17%). Hx [median (IQR) 11.1 (3.3-19.1) arbitrary units] was measured at 6.0 (2.6-11.5) years post-transplant. RTRs with high Hx (group B) had significantly higher urinary protein excretion (UP) and diastolic blood pressure than group A, despite significantly more prevalent use of renin-angiotensin-aldosterone system inhibitors. After follow-up [4.6 (3.8-5.2) years], incidence of graft failure in group A was 25 (5%) and in group B 14 (15%,P = 0.0009) After adjustment for high-sensitivity C-reactive protein (hsCRP), UP and other potential confounders, Hx remained an independent predictor of graft failure [HR = 2.5 (95% CI 1.2-5.3), P = 0.01]. In conclusion, elevated Hx predicts late graft failure in RTRs, independent of hsCRP and UP. This suggests that Hx measurement, next to measurement of creatinine clearance and UP, could be of value for the identification of RTRs at risk for graft failure.
Assuntos
Rejeição de Enxerto/epidemiologia , Rejeição de Enxerto/imunologia , Hemopexina/imunologia , Transplante de Rim/imunologia , Transplante de Rim/estatística & dados numéricos , Adulto , Doença Crônica , Creatinina/sangue , Feminino , Sobrevivência de Enxerto/imunologia , Hemopexina/metabolismo , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Nefrite/epidemiologia , Nefrite/imunologia , Valor Preditivo dos Testes , Prevalência , Estudos Prospectivos , Fatores de Risco , Fatores de TempoRESUMO
The expression of ENTPD1 (ecto-nucleoside triphosphate diphosphohydrolase) along the glomerular microvasculature of the kidney is downregulated in ischemic conditions, in contrast to E5NT (ecto-5'-nucleotidase), which may explain the increased tendency for intraglomerular microthrombus formation in vivo. It has been suggested that in ischemia, reactive oxygen species (ROS) affect glomerular ENTPD1, whereas E5NT seems less sensitive to oxidant stress. To test this hypothesis, a soluble ATP and ADP hydrolyzing enzyme solution (apyrase) [0.4 U/ml] or 5'-nucleotidase solution [0.33 U/ml] as well renal tissue were exposed to ROS, generated by gamma-irradiation in vitro. The enzymes diluted in distilled water or cryostat rat kidney sections were exposed to gamma-irradiation (0.037 Gy/s) for 0, 2, 5, 10, or 15 min, with or without supplementation of the ROS scavenger dimethylsulfoxide (DMSO). The enzyme activity of the samples was biochemically tested using standard methods, before and after irradiation. The reaction product of irradiated versus nonirradiated kidney sections was semiquantitatively evaluated after histochemical staining for either glomerular ENTPD1 or glomerular E5NT expression. The results show that the enzyme activity in samples of soluble apyrase was significantly decreased after irradiation. This effect was inhibited by DMSO. In contrast, 5'-nucleotidase activity showed only a limited decline of the activity curve after irradiation, which could also be restored following supplementation of DMSO. Glomerular ENTPD1 expression showed significant decrease after irradiation of kidney sections; again, this was inhibitable by DMSO. Glomerular E5NT activity was not altered by irradiation and DMSO supplementation did not affect its activity. It is concluded that soluble apyrase as well as the glomerular ENTPD1 are sensitive to oxidant stress, which may explain their downregulation in the ischemic condition in vivo. However, soluble 5'-nucleotidase and E5NT seem much less sensitive to ROS. This relative insensitivity of E5NT to oxidant injury may counteract ischemic injury by promoting local generation of adenosine in the ischemic micro-environment.
Assuntos
5'-Nucleotidase/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Isquemia/enzimologia , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/enzimologia , Estresse Oxidativo/fisiologia , Animais , Dimetil Sulfóxido/farmacologia , Feminino , Sequestradores de Radicais Livres/farmacologia , Raios gama , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Técnicas In Vitro , Glomérulos Renais/efeitos da radiação , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
Hemopexin is an abundant plasma protein that effectively scavenges heme. When infused into rats, hemopexin induces reversible proteinuria, and activated hemopexin is increased in children with minimal change nephrotic syndrome. These observations suggest a role for hemopexin in glomerular disease; in this study, the effects of active hemopexin on human podocytes and glomerular endothelial cells, the two cell types that compose the glomerular filtration barrier, were investigated. Within 30 min of treatment with hemopexin, actin reorganized from stress fibers to cytoplasmic aggregates and membrane ruffles in wild-type podocytes. This did not occur in nephrin-deficient podocytes unless they were transfected with nephrin-expressing plasmids. Furthermore, hemopexin did not affect actin organization in cells that do not express nephrin, specifically human glomerular endothelial cells, fibroblasts, and HEK293 cells. The effects of hemopexin on wild-type podocytes reversed within 4 h and were inhibited by preincubation with human plasma. Treatment with hemopexin activated protein kinase B in both wild-type and nephrin-deficient podocytes but activated RhoA only in wild-type cells. In addition, hemopexin led to a selective increase in the passage of albumin across monolayers of glomerular endothelial cells and to a reduction in glycocalyx. In summary, active hemopexin causes nephrin-dependent remodeling of podocytes and affects permeability of the glomerular filtration barrier by degrading the glycocalyx.
Assuntos
Actinas/metabolismo , Hemopexina/farmacologia , Proteínas de Membrana/metabolismo , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Sequência de Bases , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Glicocálix/efeitos dos fármacos , Glicocálix/metabolismo , Proteínas de Fluorescência Verde/genética , Hemopexina/genética , Hemopexina/metabolismo , Humanos , Glomérulos Renais/citologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Podócitos/ultraestrutura , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transfecção , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: Human cytomegalovirus (CMV) uses different strategies to escape from human host defense reactions. Previously we have observed that infection of endothelial cells with CMV in vitro leads to enhanced activity of endothelial ectonucleotidases. These ectoenzymes are responsible for hydrolysis of extracellular adenine nucleotides, resulting in the formation of adenosine. Infection with CMV in vivo therefore may result in local increase of adenosine production, providing an anti-inflammatory and antiaggregatory microenvironment, which may facilitate entry of the virus into the target cell. METHODS: The present study focuses on the expression of P2 type purinergic receptors on endothelial cells after infection with CMV. Human endothelial cells were infected with CMV and compared with either uninfected cells or endothelial cells infected with other herpesviruses (herpes simplex virus [HSV] 1 or 2) for the expression of P2 receptors such as P2Y1, P2Y2, or P2X7. For comparison, cells stimulated with nonspecific agents were also studied. RESULTS: A strong upregulation of the P2 receptors tested was shown, exclusively in CMV-infected cells. Stimulation with either HSV-1 or HSV2, nonspecific stimulants, or various cytokines did not affect the expression of these P2 receptors significantly. CONCLUSION: Infection of endothelium with CMV causes significant upregulation of the P2 receptors studied. As these receptors may potentially be able to concentrate nucleotides along the ectonucleotidases of the endothelial cell membrane, rapid local hydrolysis of adenosine triphosphate and adenosine diphosphate may be facilitated by enhanced P2 receptor expression. Such a CMV induced mechanism might enable the virus to escape from an important host defense response, such as local microthrombus formation.
Assuntos
Citomegalovirus/patogenicidade , Endotélio Vascular/virologia , Receptores Purinérgicos/genética , Regulação da Expressão Gênica , Humanos , Simplexvirus/patogenicidade , Cordão Umbilical/virologia , Veias UmbilicaisRESUMO
OBJECTIVE: Plasma hemopexin activity, associated with increased vascular permeability, was evaluated in healthy pregnant and non-pregnant women and in pre-eclamptic women. METHODS: Hemopexin activity and the hemopexin inhibitor, extracellular ATP, were assayed in plasma from pregnant (n = 10), preeclamptic (n = 9), and non-pregnant women (n = 10) using standard methods. Abdominal fascia tissue fragments from preeclamptic and pregnant women were immunohistochemically stained for vascular ecto-apyrase or ecto-5'nucleotidase. RESULTS: The data show significantly enhanced Hx activity exclusively in plasma from pregnant women and significantly enhanced plasma ATP in pre-eclamptic women compared with the other groups. Dephosphorylation of preeclamptic plasma resulted in reactivation of Hx activity. Fascia tissue-samples from preeclamptic women showed reduced ecto-apyrase activity and enhanced ecto-5'nucleotidase activity compared to pregnant women. CONCLUSION: Enhanced hemopexin activity may be associated with normal pregnancy, but not with preeclampsia. Decreased hemopexin in pre-eclamptic patients may be due to enhanced plasma ATP, which is possibly promoted by diminished activity of vascular ecto-apyrase.
Assuntos
Hemopexina/metabolismo , Pré-Eclâmpsia/metabolismo , 5'-Nucleotidase/metabolismo , Trifosfato de Adenosina/metabolismo , Adulto , Animais , Antígenos CD/metabolismo , Apirase/metabolismo , Biomarcadores/sangue , Biópsia , Permeabilidade Capilar , Estudos de Casos e Controles , Cesárea , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Fáscia/patologia , Feminino , Mesângio Glomerular/metabolismo , Mesângio Glomerular/fisiopatologia , Humanos , Imuno-Histoquímica , Países Baixos , Circulação Placentária , Pré-Eclâmpsia/patologia , Pré-Eclâmpsia/fisiopatologia , Gravidez , RatosRESUMO
Endothelial as well as mesangial cells show enhanced activity of ecto-apyrase following pro-inflammatory stimulation in vitro. Since this ecto-enzyme appears to be able to regulate plasma hemopexin, which latter molecule plays a role in the pathogenesis of corticosteroid responsive nephrotic syndrome, the question was raised whether glucocorticoids are potentially able to downregulate ecto-apyrase activity of these cells. Therefore, cell cultures of endothelial or mesangial were stimulated with or without lipopolysaccharide (10 ng/ml). Parallel cultures were supplemented with prednisolone with or without the glucocorticoid receptor antagonist mifepristone in various concentrations. After 24 h, cytospins were prepared and cytochemically stained for ecto-apyrase activity. mRNA for apyrase of these cells was detected using reverse transcription-polymerase chain reaction (RT-PCR). Apyrase activity of either cells or soluble apyrase (0.16 U/ml buffer) with or without supplementation of prednisolone were biochemically assayed for their phosphatase activity. The results show significantly decreased ecto-apyrase activity of lipopolysaccharide-stimulated cells after treatment with prednisolone as compared to non-prednisolone-treated cells. Preincubation with mifepristone did not inhibit the effect of prednisolone. Identical mRNA signals for apyrase were found in prednisolone and non-prednisolone-treated cells. Interestingly, soluble apyrase also showed a significant decrease of activity following preincubation with prednisolone. It is concluded that prednisolone is able to downregulate ecto-apyrase of stimulated endothelial or mesangial cells, which may potentially inhibit the conversion of hemopexin to its pro-inflammatory isoform. As blocking of the cytosolic glucocorticoid receptor showed no effect upon the prednisolone action, whereas prednisolone is able to affect soluble apyrase per se, it is felt that this particular action of prednisolone may (at least partly) be mediated through a non-genomic pathway.
Assuntos
Apirase/metabolismo , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Mesângio Glomerular/efeitos dos fármacos , Mesângio Glomerular/enzimologia , Glucocorticoides/farmacologia , Antígenos CD , Células Cultivadas , Regulação para Baixo/fisiologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , HumanosRESUMO
The pathogenesis of glomerular alterations and proteinuria in corticosteroid-responsive nephrotic syndrome (CRNS) is unknown. As an isoform of plasma hemopexin (Hx) with protease activity may be implicated in this disease, we have studied the inhibition of Hx by ADP and reactivation to its active form by endothelial or mesangial cells in vitro. We hypothesized that these cells might potentially be able to convert the inactivated form of Hx (Hxi) to active Hx (Hxa) in vitro, mediated by cellular ecto-ADPase. Since ecto-ADPase (CD39) is upregulated after stimulation of these cells with lipopolysaccharide (LPS) or certain cytokines, we postulated that this conversion might occur specifically after inflammatory stimulation of these cells. Human endothelial or mesangial cell cultures were incubated overnight with or without LPS (10.0 ng/ml) or TNFalpha (10.0 ng/ml), washed and subsequently incubated with Hxi (1.5 mg/ml) in serum-free conditions (Hxi was prepared by treatment of Hxa with ADP or ADP-beta-S). After 60 min, supernatants were tested for their capacity to alter glomerular extracellular matrix molecules (i.e. ecto-apyrase) in vitro using standard methods, and compared with Hxi that had not been incubated with cells. Supernatants containing Hxa (1.5 mg/ml) served as positive control. The results show significant activity in supernatants with Hxi (prepared using native ADP). However, Hxi inactivated by ADP-beta-S (which is non-hydrolyzable) could not be reactivated after contact with LPS-stimulated or unstimulated cells in vitro. As ecto-ADPase of these cells is upregulated by LPS, we believe that reactivation of Hxi to Hxa is mediated by cellular ecto-ADPase. Although the relevance of this inflammation-mediated activation mechanism of Hx in patients with CRNS requires further experimentation, our preliminary observations suggesting that this mechanism is corticosteroid dependent may support a potential role of Hxa in CRNS.
Assuntos
Células Endoteliais/fisiologia , Mesângio Glomerular/fisiologia , Hemopexina/fisiologia , Adenosina Trifosfatases/biossíntese , Adenosina Trifosfatases/imunologia , Adenosina Trifosfatases/metabolismo , Animais , Antígenos CD/biossíntese , Antígenos CD/imunologia , Antígenos CD/metabolismo , Apirase/biossíntese , Apirase/imunologia , Apirase/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Endopeptidases/metabolismo , Células Endoteliais/química , Células Endoteliais/enzimologia , Mesângio Glomerular/química , Mesângio Glomerular/enzimologia , Hemopexina/antagonistas & inibidores , Hemopexina/metabolismo , Histocitoquímica/métodos , Humanos , Glomérulos Renais/química , Glomérulos Renais/enzimologia , Glomérulos Renais/metabolismo , Lipopolissacarídeos/imunologia , Inibidores de Proteases/farmacologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Ratos , Veias Umbilicais/citologiaRESUMO
PROBLEM: Changes in the systemic immune response are found in preeclampsia. This may be related to high extracellular adenosine triphosphate (ATP) levels. The question arose whether ATP could affect immune responses in pregnancy. Previously, we investigated whether ATP affected monocyte activation and subpopulations. Here, we investigated ATP-induced changes in other immune cell populations in pregnant rats, systemically and in the kidney, an affected organ in preeclampsia. METHOD OF STUDY: Using flow cytometry or immunohistochemistry, blood and kidney leukocytes were studied in pregnant and non-pregnant rats at different intervals after ATP or saline infusion. RESULTS: Adenosine triphosphate (ATP) infusion induced increased peripheral blood non-classical monocytes and decreased T lymphocyte subsets in pregnant rats only, higher glomerular macrophage and T lymphocyte numbers in non-pregnant animals 1 day after infusion, and higher glomerular macrophage numbers in pregnant rats 6 days after infusion. CONCLUSION: Adenosine triphosphate (ATP) infusion in pregnant rats induced a pregnancy-specific inflammatory response. Increased ATP levels could potentially contribute to development of the inflammatory response of preeclampsia.
Assuntos
Trifosfato de Adenosina/farmacologia , Rim/efeitos dos fármacos , Gravidez/imunologia , Animais , Feminino , Citometria de Fluxo , Granulócitos/imunologia , Rim/imunologia , Contagem de Leucócitos , Macrófagos/imunologia , Monócitos/imunologia , Ratos , Ratos Wistar , Subpopulações de Linfócitos T/imunologiaRESUMO
This brief review focuses on the functional activities of plasma hemopexin recently recognized by several authors. In particular, the protease-like activity of hemopexin in vitro is linked with downregulation of the vascular angiotensin II receptor in vivo, leading to vascular expansion. Also a potential mechanism of inhibition of hemopexin activity by extracellular adenosine triphosphate is considered.
Assuntos
Angiotensina II/sangue , Hemopexina/fisiologia , Vasoconstrição/fisiologia , Vasodilatação/fisiologia , Animais , Feminino , Humanos , Masculino , Gravidez , Ligação Proteica/fisiologiaRESUMO
BACKGROUND: Hemopexin, an acute phase protein, can downregulate the angiotensin (ang) II type 1 receptor (AT1-R) in vitro. Whether hemopexin is involved in the responsiveness to ang II in vivo is unknown. Therefore, we tested whether variations in endogenous hemopexin activity are associated with the responsiveness of blood pressure to ang II in healthy volunteers. METHOD: Healthy men (nâ=â33, age 26â±â9) were studied in balance on low sodium (50âmmol Na per 24âh) and high sodium (200âmmol Na per 24âh) diet, respectively. After baseline measurements of blood pressure, ang II was infused at 0.3, 1 and 3âng/kg per min for 1âh per dose. Hemopexin activity was measured at baseline in EDTA-plasma samples by an amidolytic assay with a chromogenic substrate suitable for hemopexin activity evaluation. RESULTS: During high sodium the hemopexin activity was lower; 1.6â×â10 (0.6â×â10â-â4.7â×â10) versus 2.8â×â10 (1.1â×â10â-â5.1â×â10) arbitrary units (Pâ<â0.01) and the pressor response to 3âng ang II/kg per minute larger than during low sodium (17.6â±â6.5 versus 14.6â±â6.9âmmHg, Pâ<â0.01). Hemopexin activity negatively correlated with the pressor response to ang II during either type of sodium intake (high sodium: râ=â0.42, Pâ<â0.05; low sodium: râ=â0.35, Pâ<â0.05). CONCLUSION: These in-vivo data obtained in healthy individuals support recent in-vitro data showing that active hemopexin downregulates the availability of the AT1-R. Therefore, activated hemopexin might be considered as a factor mediating ang II effects upon blood pressure by modulating AT1-R availability.
Assuntos
Angiotensina II/fisiologia , Hemopexina/fisiologia , Adolescente , Adulto , Humanos , Masculino , Adulto JovemRESUMO
INTRODUCTION: Both nonclassical and intermediate monocytes have been implicated in different inflammatory conditions. We hypothesized that these monocytes would increase during pregnancy, a condition associated with generalized activation of inflammatory responses and that they would increase even more during preeclampsia, in which inflammatory responses are further stimulated. In the present study we investigated changes in monocyte subsets during healthy pregnancy and preeclampsia in humans and rats. METHODS: Blood monocyte subsets of nonpregnant, preeclamptic and healthy pregnant women were identified with CD14 and CD16. In nonpregnant and pregnant rats, blood monocytes were identified with CD172a and CD43, as well as in rats infused with adenosine triphosphate (ATP), a pro-inflammatory stimulus known to induce preeclampsia-like symptoms. Total and CD206-positive macrophages were quantified in placentas of these animals. RESULTS: Lower percentages of classical monocytes were found in pregnant women (91%-[83-98%]) compared to nonpregnant women (94%-[90-98%]) and even less in preeclamptic patients (90%-[61-92%]). In contrast, the percentage of combined nonclassical/intermediate monocytes was higher in pregnant women (8.5%-[2.3-16.6%] vs. 5.6%-[1.9-9.5%]) and even higher in preeclamptic patients (9.9%-[7.8-38.7%]), which was caused by a selective increase of intermediate monocytes. In rats, we also found lower percentages of classical monocytes and higher percentages of nonclassical monocytes in pregnant versus nonpregnant rats. ATP infusion increased the percentage of nonclassical monocytes in pregnant rats even further but not in nonpregnant rats. These nonclassical monocytes showed a more activated phenotype in pregnant ATP-infused rats only. Mesometrial triangles of ATP-infused rats had less CD206-positive macrophages as compared to those of saline-infused rats. CONCLUSION: The higher percentage of nonclassical/intermediate monocytes found in pregnancy and preeclampsia confirms their association with inflammatory responses. The observation that ATP stimulated numbers/activation of nonclassical monocytes in pregnant rats only, suggests that nonclassical monocytes are specifically altered in pregnancy and may play a role in the pathophysiology of preeclampsia.
Assuntos
Monócitos/classificação , Monócitos/patologia , Placenta/patologia , Pré-Eclâmpsia/patologia , Trifosfato de Adenosina , Adulto , Animais , Antígenos CD/imunologia , Feminino , Citometria de Fluxo , Humanos , Contagem de Leucócitos , Monócitos/imunologia , Placenta/imunologia , Pré-Eclâmpsia/induzido quimicamente , Pré-Eclâmpsia/imunologia , Gravidez , RatosRESUMO
The main manifestations of nephrotic syndrome include proteinuria, hypoalbuminemia, edema, hyperlipidemia and lipiduria. Common causes of nephrotic syndrome are diabetic nephropathy, minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS) and membranous nephropathy. Among the primary glomerular diseases, MCD is usually sensitive to glucocorticoid treatment, whereas the other diseases show variable responses. Despite the identification of key structural proteins in the glomerular capillary loop which may contribute to defects in ultrafiltration, many of the disease mechanisms of nephrotic syndrome remain unresolved. In this study, we show that the glomerular expression of angiopoietin-like-4 (Angptl4), a secreted glycoprotein, is glucocorticoid sensitive and is highly upregulated in the serum and in podocytes in experimental models of MCD and in the human disease. Podocyte-specific transgenic overexpression of Angptl4 (NPHS2-Angptl4) in rats induced nephrotic-range, and selective, proteinuria (over 500-fold increase in albuminuria), loss of glomerular basement membrane (GBM) charge and foot process effacement, whereas transgenic expression specifically in the adipose tissue (aP2-Angptl4) resulted in increased circulating Angptl4, but no proteinuria. Angptl4(-/-) mice that were injected with lipopolysaccharide (LPS) or nephritogenic antisera developed markedly less proteinuria than did control mice. Angptl4 secreted from podocytes in some forms of nephrotic syndrome lacks normal sialylation. When we fed the sialic acid precursor N-acetyl-D-mannosamine (ManNAc) to NPHS2-Angptl4 transgenic rats it increased the sialylation of Angptl4 and decreased albuminuria by more than 40%. These results suggest that podocyte-secreted Angptl4 has a key role in nephrotic syndrome.
Assuntos
Angiopoietinas/genética , Síndrome Nefrótica/urina , Proteinúria/prevenção & controle , Albuminúria/prevenção & controle , Proteína 4 Semelhante a Angiopoietina , Angiopoietinas/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Síndrome Nefrótica/fisiopatologia , Proteinúria/genética , Ratos , Ratos TransgênicosAssuntos
Trifosfato de Adenosina/sangue , Adenosina/sangue , Biomarcadores/sangue , Pré-Eclâmpsia/sangue , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Biomarcadores/metabolismo , Pressão Sanguínea/fisiologia , Feminino , Humanos , Modelos Cardiovasculares , Pré-Eclâmpsia/diagnóstico , Pré-Eclâmpsia/fisiopatologia , GravidezRESUMO
During normal pregnancy, in contrast to preeclampsia, plasma hemopexin activity is increased together with a decreased vascular angiotensin II receptor (AT(1)) expression. We now tested the hypothesis that hemopexin can downregulate the AT(1) receptor in vitro. Analysis of human monocytes or endothelial cells by flow cytometry showed decreased membrane density of AT(1) exclusively after incubation with active hemopexin, whereas in supernatants of these cell cultures, AT(1) molecules could be detected (dot blotting). Also, diminished AT(1) was observed in endothelial cell lysates after contact with hemopexin (Western blotting). Hemopexin also induced extracellular signal-regulated kinase 1/2 pathway inhibition in cells after stimulation with angiotensin II in vitro, indicating downregulation of AT(1) by hemopexin. In addition, functional loss of AT(1) occurred after incubation of rat aortic rings with active hemopexin, as reflected by decreased contraction of the aortic rings on stimulation with angiotensin II. It was further demonstrated that plasma from normal pregnant women decreased the AT(1) receptor expression on monocytes as compared with plasma from nonpregnant women or preeclamptic women. Finally, it was shown that plasma hemopexin activity increases during normal gestation from week 10 onward. We concluded that active hemopexin is able to downregulate the AT(1) receptor in human monocytes, endothelial cells, and rat aortic rings. We propose that the physiological role of enhanced hemopexin activity during healthy pregnancy is to downregulate the vascular AT(1) receptor, promoting an expanded vascular bed. Inhibition of hemopexin activity during preeclampsia may result in an enhanced AT(1) receptor expression and a contracted vascular bed.
Assuntos
Endotélio Vascular/citologia , Hemopexina/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/metabolismo , Análise de Variância , Western Blotting , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Endotélio Vascular/efeitos dos fármacos , Feminino , Humanos , Técnicas In Vitro , Leucócitos Mononucleares/fisiologia , Músculo Liso Vascular/fisiologia , Pré-Eclâmpsia/sangue , Gravidez , Probabilidade , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Valores de Referência , Fatores de Risco , Sensibilidade e Especificidade , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologiaRESUMO
Since an active isoform of plasma hemopexin (Hx) has been proposed to be a potential effector molecule in minimal change disease (MCD), we tested plasma and urine samples from subjects with MCD in relapse (n = 18) or in remission (n = 23) (after treatment with prednisolone) for presence or activity of Hx. For comparison, plasma or urine from proteinuric subjects with focal and segmental glomerulosclerosis (FSGS, n = 11), membranoproliferative glomerulonephritis (MPGN, n = 9), IgA nephropathy (n = 5) or healthy control donors (n = 10), were incorporated into the study. Electrophoresis and Western blotting methods were used for evaluation of the Hx status, whereas protease activity of Hx was tested upon kidney tissue in vitro according to standard methods. The results show (1) a decreased mean titer of plasma Hx exclusively in MCD relapse subjects as compared with MCD in remission (0.21+/-0.14 mg/ml vs 0.44+/-0.06 mg/ml; p < 0.01). Mean Hx titers in other proteinuric subjects ranged from 0.38+/-0.05 mg/ml to 0.40+/- 0.06 mg/ml, whereas, the mean titer of healthy controls was 0.59+/-0.03 mg Hx/ml; (2) an increased Hx activity (expressed in arbitrary units) exclusively in plasma from MCD relapse subjects (3.3+/-0.72 vs 1.16+/-0.56, MCD remission; p < 0.01); (3) different Western blot patterns in MCD relapse vs remission plasma; (4) reduced stainability or virtual absence of the 80-kD Hx band in blots of urine from MCD relapse in contrast to urine samples from other proteinuric subjects with FSGS, MPGN, or IgA nephropathy. It is concluded that Hx in MCD relapse subjects may exist in an altered isoform, showing enhanced protease activity as compared with subjects in remission, subjects with other forms of primary glomerulopathy, or healthy control individuals.
Assuntos
Hemopexina/metabolismo , Nefrose Lipoide/sangue , Adolescente , Western Blotting , Estudos de Casos e Controles , Criança , Eletroforese , Feminino , Glomerulonefrite por IGA/urina , Glomerulonefrite Membranoproliferativa/urina , Glomerulosclerose Segmentar e Focal/urina , Hemopexina/urina , Humanos , Masculino , Peptídeo Hidrolases/sangue , Proteinúria/sangue , Proteinúria/etiologia , Proteinúria/urina , Recidiva , Indução de RemissãoRESUMO
BACKGROUND: Previous studies into the relevance of a putative circulating factor in the pathogenesis of minimal change nephrotic syndrome have opened the possibility that plasma hemopexin might be an important effector molecule in this disorder. Thus, intra renal infusion of isolated plasma hemopexin into rats induced minimal change like glomerular lesions and proteinuria. Both, in vivo and in vitro effects of the active isoform of hemopexin could be attributed to protease activity of this molecule. However, the question remained whether hemopexin per se rather than some contaminating plasma factor is responsible for the potential enzymatic activity of this molecule. METHODS: Recombinant hemopexin was prepared according to standard methods in Pichia pastoris and compared for its identity and protease activity with plasma hemopexin using Western blotting and various in vitro assays. Unilateral renal perfusion in anesthetized rats was used to test the proteinuria inducing capacity of recombinant hemopexin versus heat-inactivated recombinant hemopexin. RESULTS: The blotting results show identical 85 kD bands in both native as well as recombinant hemopexin. Incubation of kidney tissue with recombinant hemopexin resulted in loss of of glomerular ectoapyrase and sialoglycoproteins, as shown by immunohistochemistry, which effect can be inhibited with the serine protease inhibitor phenylmethanesulfonyl fluoride. Artificial substrates for serine proteases, like kallikrein or thrombin, are hydrolysed by recombinant hemopexin in vitro, and not by heat-inactivated recombinant hemopexin or saline. Unilateral kidney perfusion of recombinant hemopexin, in contrast to control Pichia transfection products or heat-inactivated recombinant hemopexin, followed by a protein marker showed significantly enhanced urinary protein leakage 5.0, 10.0, and 15.0 minutes after perfusion. CONCLUSION: It is concluded that the hemopexin molecule as such can potentially act as a toxic protease, leading in the rat to proteinuria and glomerular alterations characteristic for minimal change nephrotic syndrome.
Assuntos
Hemopexina/genética , Hemopexina/metabolismo , Nefrose Lipoide/metabolismo , Nefrose Lipoide/fisiopatologia , Peptídeo Hidrolases/metabolismo , Animais , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Feminino , Taxa de Filtração Glomerular , Humanos , Neoplasias Hepáticas , Pichia/genética , Proteinúria/metabolismo , Proteinúria/fisiopatologia , Ratos , Ratos Wistar , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Ischemic injury plays an important role in chronic renal transplant failure (CRTF). Down-regulation of ecto-adenosine triphosphatase (ATPase) in combination with up-regulation of ecto-5'-nucleotidase is a hallmark of ischemic injury. We studied the expression of renal ecto-5'-nucleotidase and ecto-ATPase in experimental renal transplantation. Fisher 344-to-Lewis allografted rats were either treated with an angiotensin-converting enzyme inhibitor (ACEi) or left untreated. Lewis-to-Lewis syngrafted rats served as controls. Untreated allografted rats developed proteinuria, glomerulosclerosis, and mild intimal hyperplasia. ACEi completely prevented focal and segmental glomerulosclerosis (FGS) and proteinuria, but significantly enhanced intimal hyperplasia. Untreated allografted rats revealed marked vascular ecto-5'-nucleotidase activity, which increased with ACEi. Vascular ecto-5'-nucleotidase activity was absent in syngrafted animals. Ecto-5'-nucleotidase activity correlated well with intimal hyperplasia. Glomerular ecto-ATPase expression was significantly reduced in untreated allografted rats compared to syngrafted rats and correlated well with the extent of FGS. ACEi prevented reduction in glomerular ecto-ATPase. We found de-novo expression of ecto-5'-nucleotidase at sites of renal intimal hyperplasia. Glomerular ecto-ATPase expression was markedly reduced in allografted rats and was prevented by ACEi. These enzyme expression patterns suggest local ischemic damage in experimental CRTF.
Assuntos
5'-Nucleotidase/genética , Adenosina Trifosfatases/genética , Glomérulos Renais/patologia , Transplante de Rim/patologia , Animais , Imuno-Histoquímica , Glomérulos Renais/enzimologia , Masculino , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Transplante Homólogo , Falha de TratamentoRESUMO
INTRODUCTION: Chronic allograft nephropathy (CAN) is a major cause of deterioration of kidney function in transplanted patients. It is thought that glomerular ischaemia may contribute to glomerular dysfunction and proteinuria in these subjects. As reduced expression of glomerular ecto-ATPase concurrent with upregulation of glomerular ecto-AMPase activity is associated with local ischaemia, we compared the expression of these glomerular ecto-enzymes in kidney biopsies from subjects with CAN or with acute rejection vs normal human kidney tissue. METHODS: Kidney biopsies in comparison with normal kidney tissue samples (n = 10) were studied from subjects with CAN (n = 6), acute interstitial rejection (n = 13), acute vascular rejection (n = 3), or subjects whose biopsies were histologically difficult to classify (n = 8). Cryostat sections (4 micro m) were stained for ecto-ATPase using standard procedures. For the demonstration of ecto-AMPase activity, conventional enzyme histochemistry was used. Reaction product of individual sections was quantified using computerized image analysis. RESULTS: The results clearly show decreased expression of glomerular ecto-ATPase in combination with increased glomerular ecto-AMPase in all biopsies from subjects with CAN vs normal kidney tissue (P < 0.001). Although to a lesser extent, this staining pattern was also observed in patients with vascular rejection as well as in subjects whose biopsies were histologically difficult to classify (P < 0.01), while biopsies from subjects with interstitial rejection and normal control tissue stained negative for glomerular ecto-AMPase. CONCLUSION: In CAN diminished glomerular ecto-ATPase expression occurs in association with significantly enhanced activity of glomerular ecto-AMPase. This is a strong indication for ischaemic injury of the glomerular microvasculature. As positive staining for ecto-AMPase in acute rejection episodes may be an important sign for long-term prognosis, we feel that screening of biopsies from individual subjects for glomerular ecto-AMPase activity should be considered.