RESUMO
Genetic polymorphism studies of cytokines may provide an insight into the understanding of acute kidney injury (AKI) and death in intensive care unit (ICU) patients. The aim of this study was to investigate whether the genetic polymorphisms of -308 G < A tumour necrosis factor (TNF)-α, -174 G > C interleukin (IL)-6 and -1082 G > A IL-10 may predispose ICU patients to the development of AKI and/or death. In a prospective nested case-control study, 303 ICU patients and 244 healthy individuals were evaluated. The study group included ICU patients who developed AKI (n = 139) and 164 ICU patients without AKI. The GG genotype of TNF-α (low producer phenotype) was significantly lower in the with AKI than without AKI groups and healthy individuals (55 versus 62 versus 73%, respectively; P = 0·01). When genotypes were stratified into four categories of TNF-α/IL-10 combinations, it was observed that low TNF-α plus low IL-10 producer phenotypes were more prevalent in patients with AKI, renal replacement therapy and death (P < 0·05). In logistic regression analysis, low TNF-α producer plus low IL-10 producer phenotypes remained as independent risk factors for AKI and/or death [odds ratio (OR) = 2·37, 95% confidence interval (CI): 1·16-4·84; P = 0·02] and for renal replacement therapy (RRT) and/or death (OR = 3·82, 95% CI: 1·19-12·23; P = 0·02). In this study, the combination of low TNF-α plus low IL-10 producer phenotypes was an independent risk factor to AKI and/or death and RRT and/or death in critically ill patients. Our results should be validated in a larger prospective study with long-term follow-up to emphasize the combination of these genotypes as potential risk factors to AKI in critically ill patients.
Assuntos
Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/imunologia , Interleucina-10/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Injúria Renal Aguda/genética , Injúria Renal Aguda/mortalidade , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Análise Mutacional de DNA , Feminino , Predisposição Genética para Doença , Humanos , Unidades de Terapia Intensiva , Interleucina-10/genética , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Prognóstico , Estudos Prospectivos , Análise de Sobrevida , Fator de Necrose Tumoral alfa/genéticaRESUMO
In this study, we compared the properties of a serine endopeptidase H1 (SH1) and a serine thiol endopeptidase (STH2) purified from human urine by DEAE-cellulose followed by a Bio Gel A0.5 m or Sepharose Mercurial chromatographs. These enzymes differ in their action upon different hormone peptides. We used fluorogenic substrates to further characterize the enzyme. The substrate specificity of urinary SH1 was studied using different internally quenched fluorescent peptides, and AbzFGQEDDnp was hydrolyzed by SH1. Other enzymes present in urine, such as serine endopeptidase H2, prolyl endopeptidase, neutral endopeptidase like and angiotensin-I converting enzyme, were not able to hydrolyze this substrate. SH1 is 100% inhibited by PMSF and resistant to EDTA, OPA, thiorphan, E64, pOHMB and phosphoramidon. Endopeptidase STH2 is completely inhibited by PMSF, E64 and pOHMB. Enzyme SH1 hydrolyzes the peptide bound F5-S6 at bradykinin (BK: RPPGFSPFR) molecule and R-Q at AbzBKQEDDnp. When studying enzyme STH2, the cleavage sites determined to the related substrates were F5-S6 using BK as substrate and F-R using AbzBKQEDDnp. The kilometers value obtained for AbzBKQEDDnp and AbzFGQEDDnp were 1.18 and 0.007 uM, respectively. Kininases from kidney and urine can hydrolyze peptide bounds from components of the kallikrein-kinin system, the angiotensin-renin system and the neuropeptides system, straight contributing in kidney homeostasis. SH1 was located at the distal tubule [Casarini et al., 1999a, Am. J. Physiol. 277, F66] and can have an important function in the control of kinin found in this portion, since is known that all components of the kallikrein-kinin system were found in this portion. The physiological role of SHT2 could be related to the inter-relation between the kallikrein-kinin system and neuropeptides in the control of the water electrolyte balance [Braz. J. Med. Biol. Res. 25 (3) (1992) 219].
Assuntos
Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/urina , Sequência de Aminoácidos , Humanos , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Serina Endopeptidases/metabolismo , Especificidade por SubstratoRESUMO
A prolyl endopeptidase (PE) was purified 83 times from human urine by DEAE-cellulose and Sepharose Mercurial chromatographies. In this work we studied the specificity of PE using different fluorogenics substrates. Further characterization of the enzyme was carried out using BK and it's analogue, Abz-RPPGFSPFRQ-EDDnp and Abz-FPQ-EDDnp, for measure of enzymatic activity of prolyl endopeptidase (Abz=ortho-aminobenzoic acid; EDDnp=N-[2, 4-dinitrophenyl]ethylenediamine). The substrate Abz-FPQ-EDDnp was considered as specific for PE. The endopeptidase PE, with a molecular weight of 45 kDa, was inhibited 100% by EDTA and pOHMB and resistant to PMSF, thyorphan, E64 and phosphoramidon, when we used the mentioned substrates. These results suggest that PE is a metallo endopeptidase that contains a thiol group important for it's activity. It was also able to hydrolyze in Abz-RPPGFSPFRQ-EDDnp the F-R peptide bound, differing from those obtained upon BK molecule, where the enzyme prefer the peptide bound located after double proline. In the substrate Abz-FPQ-EDDnp PE hydrolyzes the P-Q peptide bound. Furthermore the urinary PE is particularly unable to hydrolyze peptides with single prolines such as substance P, neurotensin and LHRH. The determined K(m) for Abz-RPPGFSPFRQ-EDDnp and Abz-FPQ-EDDnp were 0.74 and 0.65 uM, respectively. The optimum pH for the PE activity, using the substrate Abz-RPPGFSPFRQ-EDDnp was approximately 9.0, but using the specific substrate Abz-FPQ-EDDnp was 6.5 and 8.0. Endopeptidases, which are situated at brush border surface from proximal tubules, have an important role in kidney handling of many peptides, which are filtered by the glomerulus. The prolyl endopeptidase located at distal tubule could have an important physiological function in control of kinin formed in this portion. It's known that all components from kallicrein-kinin system like low molecular weigh kininogen and kallikrein are presents in this portion.
Assuntos
Serina Endopeptidases/urina , Bradicinina/análogos & derivados , Bradicinina/metabolismo , Cromatografia em Agarose , Cromatografia DEAE-Celulose , Cromatografia Líquida de Alta Pressão , Corantes Fluorescentes/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Oligopeptídeos/metabolismo , Prolil Oligopeptidases , Inibidores de Proteases/farmacologia , Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/metabolismo , Especificidade por SubstratoRESUMO
We have previously described a kinin-inactivating endopeptidase (H2), which was purified 19-fold from human urine by DEAE-cellulose chromatography and gel filtration. The enzyme was inhibited 100% by PMSF, TPCK and pOHMB. In the present communication, we further characterized this enzyme using the fluorogenic substrates Abz-RPPGFSPFRQ-EDDnp (Abz-BKQ-EDDnp) and Abz-FRQ-EDDnp (Abz = ortho-aminobenzoic acid; EDDnp = N-[2,4-dinitrophenyl] ethylenediamine). Also a rapid, sensitive and specific assay for the H2 was developed. The enzyme hydrolyzed bradykinin (BK = RPPGFSPFR) at the F-S peptide bond, differing from the cleavage site F-R, in the fluorogenic substrates Abz-BKQ-EDDnp and Abz-FRQ-EDDnp. Other enzymes present in urine as the serine endopeptidase H1, prolyl endopeptidase and neutral endopeptidase-like were not able to hydrolyze the related substrate Abz-FRQ-EDDnp. The determined Km for Abz-BKQ-EDDnp and Abz-FRQ-EDDnp were 0.79 microM and 3.02 microM, respectively. Using the fluorogenic substrates, we observed that PMSF and p-hydroxymercuribenzoate irreversibly inhibited the enzyme H2. E-64 was a weak and reversible inhibitor, whereas EDTA and pepstatin were not inhibitory. The inhibition observed in the presence of pOHMB was partially reversed by 2 mM cysteine. These results suggest that the H2 enzyme belongs to the subfamily of SH-containing serine proteases. Based on the molecular weight of isolated H2 (60 kDa), we believe that this enzyme originated from the kidney and may cleave the kinins filtered through the glomerulus and also that produced in the kidney.
Assuntos
Endopeptidases/química , Endopeptidases/urina , Cininas/metabolismo , Serina Endopeptidases , Bradicinina , Endopeptidases/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Hidrólise , Fragmentos de Peptídeos , Especificidade por SubstratoRESUMO
OBJECTIVE: The aims of this study were to purify and characterize a neutral endopeptidase-like enzyme (NEP-like) in human urine and propose a rapid, sensitive and specific assay for this enzyme using the fluorogenic substrate Abz-FDQ-EDDnp, where Abz = O-aminobenzoic acid and EDDnp = N-(2,4-dinitrophenyl)ethylenediamine. METHODS: Soluble urinary NEP was purified from human urine using a DEAE-cellulose Cellex D column and gel filtration on an AcA-44 column. NEP-like activity was assayed by its ability to hydrolyse bradykinin (BK) and the fluorogenic substrates Abz-BKQ-EDDnp and Abz-FDQ-EDDnp. The Km was determined using Abz-FDQ-EDDnp as a substrate. The hydrolysis products of BK and Abz-FDQ-EDDnp were analysed by high-performance liquid chromatography (HPLC). The mol. wt was estimated by polyacrylamide gel electrophoresis and the enzyme analysed by Western blot using the antibody obtained from purified recombinant NEP expressed in Pichia pastoris yeast. RESULTS: The NEP-like was purified from human urine until homogeneity and presented a mol. wt of 94000. The substrate Abz-FDQ-EDDnp was selectively hydrolysed at the F-D bond by NEP-like and by recombinant NEP. For this substrate, the NEP-like activity was maximal at pH 7.0, although a small peak of activity was observed at pH 8.0, and the determined Km was 14 microM. The enzymatic activity was inhibited by thiorphan and phosphoramidon. In Western blot analysis, NEP-like reacted strongly with a polyclonal antibody for NEP. CONCLUSION: A NEP-like enzyme was purified from human urine. Based on the mol. wt of the isolated NEP-like enzyme, it was concluded that this enzyme was produced in the kidney. In the kidney, this enzyme may cleave the kinins filtered through the glomerulus and also the kinins produced in the distal nephron. An internally quenched fluorogenic peptide, Abz-FDQ-EDDnp, was selectively hydrolysed by NEP-like and by recombinant NEP.
Assuntos
Neprilisina/urina , Cromatografia DEAE-Celulose , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes , Humanos , Concentração de Íons de Hidrogênio , Imunoquímica , Rim/enzimologia , Cinética , Peso Molecular , Neprilisina/antagonistas & inibidores , Neprilisina/isolamento & purificação , Especificidade por SubstratoRESUMO
OBJECTIVE: Previous analysis of the angiotensin I-converting enzyme (ACE) gene in this laboratory showed that primary mesangial cells in culture are able to express ACE mRNA. Moreover, ACE is produced as an ectoenzyme and as a secreted form of the enzyme, indicating a potential effect of local angiotensin II production on glomerular microcirculation. The aim of this study was to purify and characterize the secreted and intracellular ACE forms from mesangial cells in culture. METHODS AND RESULTS: Medium from Wistar rats mesangial cells was collected (third passage), incubated for 20 h with RPMI without fetal bovine serum and concentrated 29 times in an Amicon concentrator. The concentrated medium was submitted to gel filtration on an AcA-34 column and two peaks (ACE1, mol. wt 130 000 and ACE2, 60000) with ACE on activity Hippuryl-His-Leu and Z-Phe-His-Leu were separated. The mesangial cells were collected and ACE enzyme was extracted using Triton X-114, followed by centrifugation and concentration. The supernatant was submitted to the same chromatography as described above and two peaks with ACE activity (ACEInt1, mol. wt 130000 and ACEInt2, 68000) were separated. The purified ACE were inhibited by enalaprilat and captopril, two potent competitive inhibitors of ACE and by EDTA, using Hippuryl-His-Leu as a substrate. The Km values were 2 mM for ACE1 and ACE2 and 3 mM for ACEInt1 and ACEInt2. The enzymes ACE1 and ACE2 presented an optimum pH of 8.0 and ACEInt1 and ACEInt2 an optimum pH of 7.5. CONCLUSION: The activities of full-length wild-type and N-domain ACE were characterized by the ratio of the hydrolysis of Z-Phe-His-Leu/Hippuryl-His-Leu, which was 1 and 4, respectively. The ratios found for ACE1, ACE2, ACEInt1 and ACEInt2 in the present study were similar to those described above, suggesting that mesangial cells, besides showing the presence of intracellular ACE, are able to secret both full-length wild-type ACE and N-domain ACE. Thus, they may potentially have an effect, not only on bradykinin and angiotensin I (ACE wild-type), but also on substance P, luteinizing hormone-releasing hormone and Met-enkephalin to interfere with glomerular haemodynamics and with the renal microcirculation.