Protective effect of recombinant Schistosoma japonicum cystatin against acute kidney injury associated with acute liver failure in mice / 中国血吸虫病防治杂志

OBJECTIVE@#To evaluate the protective effect of recombinant Schistosoma japonicum cystatin (rSj-Cys) against acute kidney injury induced by acute liver failure and unravel the underlying mechanism, so as to provide insights into the clinical therapy of acute kidney injury.@*METHODS@#Twenty-four male C57BL/6J mice at ages of 6 to 8 weeks were randomly divided into the normal control group, rSj-Cys control group, lipopolysaccharide (LPS)/D-galactosamine (D-GaIN) model group and LPS/D-GaIN + rSj-Cys treatment group, of 6 mice each group. Mice in the LPS/D-GaIN group and LPS/D-GaIN + rSj-Cys group were intraperitoneally injected with LPS (10 μg/kg) and D-GaIN (700 mg/kg), and mice in the LPS/D-GaIN + rSj-Cys group were additionally administered with rSj-Cys (1.25 mg/kg) by intraperitoneal injection 30 min post-modeling, while mice in the rSj-Cys group were intraperitoneally injected with rSj-Cys (1.25 mg/kg), and mice in the normal control group were injected with the normal volume of PBS. All mice were sacrificed 6 h post-modeling, and mouse serum and kidney samples were collected. Serum creatinine (Cr) and urea nitrogen (BUN) levels were measured, and the pathological changes of mouse kidney specimens were examined using hematoxylin-eosin (HE) staining. Serum tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels were detected using enzyme-linked immunosorbent assay (ELISA), and the expression of inflammatory factors and pyroptosis-related proteins was quantified in mouse kidney specimens using immunohistochemistry. In addition, the expression of pyroptosis-related proteins and nuclear factor-kappa B (NF-κB) signaling pathway-associated proteins was determined in mouse kidney specimens using Western blotting assay.@*RESULTS@#HE staining showed no remarkable abnormality in the mouse kidney structure in the normal control group and the rSj-Cys control group, and renal tubular injury was found in LPS/D-GaIN group, while the renal tubular injury was alleviated in LPS/D-GaIN+rSj-Cys treatment group. There were significant differences in serum levels of Cr (F = 46.33, P < 0.001), BUN (F = 128.60, P < 0.001), TNF-α (F = 102.00, P < 0.001) and IL-6 (F = 202.10, P < 0.001) among the four groups, and lower serum Cr [(85.35 ± 32.05) μmol/L], BUN [(11.90 ± 2.76) mmol/L], TNF-α [(158.27 ± 15.83) pg/mL] and IL-6 levels [(56.72 ± 4.37) pg/mL] were detected in the in LPS/D-GaIN + rSj-Cys group than in the LPS/D-GaIN group (all P values < 0.01). Immunohistochemical staining detected significant differences in TNF-α (F = 24.16, P < 0.001) and IL-10 (F = 15.07, P < 0.01) expression among the four groups, and lower TNF-α [(106.50 ± 16.57)%] and higher IL-10 expression [(91.83 ± 5.23)%] was detected in the LPS/D-GaIN + rSj-Cys group than in the LPS/D-GaIN group (both P values < 0.01). Western blotting and immunohistochemistry detected significant differences in the protein expression of pyroptosis-related proteins NOD-like receptor thermal protein domain associated protein 3 (NLRP3) (F = 24.57 and 30.72, both P values < 0.001), IL-1β (F = 19.24 and 22.59, both P values < 0.001) and IL-18 (F = 16.60 and 19.30, both P values < 0.001) in kidney samples among the four groups, and lower NLRP3, IL-1β and IL-18 expression was quantified in the LPS/D-GaIN + rSj-Cys treatment group than in the LPS/D-GaIN group (P values < 0.05). In addition, there were significant differences in the protein expression of NF-κB signaling pathway-associated proteins p-NF-κB p-P65/NF-κB p65 (F = 71.88, P < 0.001), Toll-like receptor (TLR)-4 (F = 45.49, P < 0.001) and p-IκB/IκB (F = 60.87, P < 0.001) in mouse kidney samples among the four groups, and lower expression of three NF-κB signaling pathway-associated proteins was determined in the LPS/D-GaIN + rSj-Cys treatment group than in the LPS/D-GaIN group (all P values < 0.01).@*CONCLUSIONS@#rSj-Cys may present a protective effect against acute kidney injury caused by acute liver failure through inhibiting inflammation and pyroptosis and downregulating the NF-κB signaling pathway.

Nueva ecuación CAPA dependiente de Cistatina-C: mayor detección de filtrado glomerular disminuido en pacientes VIH / New CAPA-dependent Cystatin C Equation: increased detection of decreased glomerular filtration in HIV-infected patients

INTRODUCCIÓN: Creatinina y sus ecuaciones presentan claras limitaciones en relación a su baja sensibilidad para identificar etapas iniciales de disfunción renal. Cistatina-c ha sido propuesta como un marcador prometedor, pero hasta ahora, no hay evidencia que demuestre la superioridad de sus ecuaciones por sobre las de creatinina. Sin embargo, no existen estudios que comparen el rendimiento de la última ecuación de cistatina desarrollada por Grubb y colaboradores en 2014, la ecuación "CAPA". OBJETIVOS: Analizar el rendimiento de CAPA para detectar disminución temprana del filtrado glomerular en pacientes VIH, en comparación con ecuaciones dependientes de creatinina: Cockroft-Gault, MDRD-4, CKD-EPI y MCQ. MATERIAL Y MÉTODOS: Estudio analítico, observacional, transversal. Realizado entre julio y noviembre de 2017, en un hospital de tercer nivel de Argentina. Incluyó pacientes VIH realizando antirretrovirales, ≥18 años. Se excluyeron casos con creatinina ≥1,2 mg/dl. RESULTADOS: Se reclutaron 100 pacientes, y se incluyeron 89: 47 (52,8%) fueron mujeres. CAPA detectó disminuciones más pronunciadas del FG que las ecuaciones dependientes de creatinina. Las medias de FG por CAPA mostraron diferencias con las medias por Cockroft-Gault (p<0,0001); MDRD-4 (p=0,005); CKD-EPI (p<0,0001) y MCQ (p<0,0001). De los 46 casos (51,7%) con FG <90ml/min detectados a través de cualquier ecuación utilizada CAPA detectó 82,6% vs. 71,7% detectados por las cuatro fórmulas de creatinina en conjunto (p<0,0001), y que cada ecuación de creatinina individualmente: CAPA vs. Cockroft-Gault (p=0,01); vs. MDRD-4 (p<0,0001); vs. CKD-EPI (p=0,005). CONCLUSIONES: CAPA detectó disminuciones más marcadas del FG que las ecuaciones dependientes de creatinina en pacientes VIH

INTRODUCTION: Creatinine and its equations have clear limitations regarding their low sensitivity to identify initial stages of renal dysfunction. Cystatin C has been proposed as a promising marker, but so far, there has been no evidence showing the superiority of its equations over the creatinine ones. However, there are no studies which compare the performance of the latest cystatin equation developed by Grubb and collaborators in 2014: the "CAPA" equation. OBJECTIVES: To analyze the performance of CAPA equation to detect early reduction of glomerular filtration in HIV-infected patients, in comparison with creatinine-dependent equations: Cockroft-Gault, MDRD-4, CKD-EPI and MCQ. METHODS: An analytical, observational, cross-sectional study was conducted between July and November 2017, at an Argentinian specialty hospital. ≥18-year old HIV-infected patients undergoing antiretroviral therapy were included. Cases with creatinine ≥1.2 mg/dL were excluded. RESULTS: 100 patients were recruited, and 89 were included: 47 (52.8%) were women. CAPA equation detected more pronounced decreases in GFR than the creatinine-dependent equations. The mean values of GFR obtained by CAPA showed differences with the ones found through Cockroft-Gault (p <0.0001); MDRD-4 (p = 0.005); CKD-EPI (p <0.0001) and MCQ (p <0.0001). Of the 46 cases (51.7%) with GFR <90 ml/min detected through the use of any equation, CAPA detected 82.6% vs. 71.7% detected by the four creatinine formulas together (p <0.0001) and by each creatinine equation individually: CAPA vs. Cockroft-Gault (p = 0.01); vs. MDRD-4 (p <0.0001); vs. CKD-EPI (p = 0.005). CONCLUSIONS: CAPA equation detected more marked decreases in GFR than the creatinine-dependent equations in HIV-infected patients

Early term effect of ureterorenoscopy (URS) on the Kidney: research measuring NGAL, KIM-1, FABP and CYS C levels in urine

ABSTRACT Aim: URS is a very commonly used procedure for treatment of ureter stones. Increased hydrostatic pressure in the collecting system linked to fluids used during the procedure may cause harmful effects on the kidney. The aim of this study is to determine whether the URS procedure has a negative effect on the kidney by investigating NGAL, KIM-1, FABP and Cys C levels in urine. Material and Methods: This study included 30 patients undergoing ureterorenoscopy (URS) for ureter stones. Urine samples were collected 5 times; before the URS procedure (control) and at 1, 3, 5 and 12 hours following the procedure. NGAL, KIM-1, FBAP and Cys C levels were measured in urine and compared with the control values. Results: The NGAL levels in urine before the procedure and at 1, 3, 5 and 12 hours after the procedure were 34.59±35.34; 62.72±142.32; 47.15±104.48; 45.23±163.16 and 44.99±60.79ng/mL, respectively (p=0.001). Similarly, the urinary KIM-1, FABP and Cys C levels were found to increase compared to control values; however this increase did not reach statistical significance (p >0.05). Conclusions: After the URS procedure, there were important changes in NGAL, FABP, KIM-1 and Cys C levels. These changes reached statistical significance for NGAL, but did not reach significance for the other parameters. In conclusion, the URS procedure significantly affects the kidney; however, this effect disappears over time.

Diagnosis, classification and treatment of gestational trophoblastic neoplasia / Diagnóstico, classificação e tratamento da neoplasia trofoblástica gestacional

Gestational trophoblastic neoplasia (GTN) is the term to describe a set of malignant placental diseases, including invasive mole, choriocarcinoma, placental site trophoblastic tumor and epithelioid trophoblastic tumor. Both invasive mole and choriocarcinoma respond well to chemotherapy, and cure rates are greater than 90%. Since the advent of chemotherapy, low-risk GTN has been treated with a single agent, usually methotrexate or actinomycin D. Cases of high-risk GTN, however, should be treated with multiagent chemotherapy, and the regimen usually selected is EMA-CO, which combines etoposide, methotrexate, actinomycin D, cyclophosphamide and vincristine. This study reviews the literature about GTN to discuss current knowledge about its diagnosis and treatment.

Neoplasia trofoblástica gestacional (NTG) é o termo que descreve o conjunto de anomalias malignas da placenta, incluindo a mola invasora, coriocarcinoma, tumor trofoblástico do sítio placentário e tumor trofoblástico epitelióide. Ambos a mola invasora e o coriocarcinoma respondem bem à quimioterapia, com taxas de cura superiores a 90%. Desde o advento da quimioterapia, NTG de baixo risco tem sido tratada com monoquimioterapia, pelo geral methotrexate ou actinomicina-D. Casos de NTG de alto risco, contudo, devem ser tratados com poliquimioterapia, e o regime usualmente escolhido é o EMA-CO que combina etoposide, methotrexate, actinomicina-D, ciclofosfamida e vincristina. Esse estudo revê a literatura sobre NTG a fim de discutir os conhecimentos atuais sobre seu diagnóstico e tratamento.

Neck circumference as a potential marker of metabolic syndrome among college students / Circunferência do pescoço como possível marcador para síndrome metabólica em universitários / La circunferencia del cuello como posible indicador del síndrome metabólico en universitarios

OBJECTIVE: to relate neck circumference with metabolic syndrome and its criteria among college students. METHOD: cross-sectional study conducted with 702 college students in Fortaleza, CE, Brazil from September 2010 to June 2011. Socio-demographic data, waist circumference and neck circumference were collected together with blood pressure, fasting blood sugar, triglyceride levels, and HDL-C. RESULTS: 1.7% of the studied sample presented metabolic syndrome. Of these, 58.3% presented altered neck circumference (p<0.006). As neck circumference decreases, pressure levels improve (p<0.001). Additionally, college students with high fasting blood sugar (p=0.003) and high triglyceride levels (p<0.001) presented higher values of neck circumference. CONCLUSION: neck circumference is a potential predictive marker in the detection of metabolic syndrome and its components among college students. .

OBJETIVO: relacionar a circunferência do pescoço com a síndrome metabólica e seus critérios em universitários. MÉTODO: estudo transversal, realizado com 702 universitários de Fortaleza, CE, Brasil, no período de setembro de 2010 a junho de 2011. Coletaram-se dados sociodemográficos, circunferência da cintura, circunferência do pescoço, níveis de pressão arterial e glicemia plasmática de jejum, triglicerídeos e lipoproteína de alta densidade. RESULTADOS: 1,7% da amostra investigada tinha a síndrome metabólica. Desses, 58,3% apresentaram circunferência do pescoço alterada (p<0,006). Na medida em que decresce a circunferência do pescoço, os valores pressóricos dos universitários melhoram (p<0,001). Também, observou-se que universitários com valores de glicemia de jejum plasmática (p=0,003) e triglicerídeos (p<0,001) elevados apresentaram maiores valores de circunferência do pescoço. CONCLUSÃO: a circunferência do pescoço mostrou-se um possível marcador preditivo para detecção da síndrome metabólica e seus componentes em universitários. .

OBJETIVO: relacionar la circunferencia del cuello con el síndrome metabólico y sus criterios en universitarios. MÉTODO: estudio transversal realizado con 702 universitarios de Fortaleza-CE, Brasil, en el período de septiembre de 2010 a junio de 2011. Se recolectaron datos sociodemográficos, circunferencia de la cintura, circunferencia del cuello, niveles de presión arterial y glucemia plasmática de ayuno, triglicéridos y HDL-C. RESULTADOS: 1,7% de la muestra investigada tenían el síndrome metabólico. De estos, 58,3% presentaron circunferencia del cuello alterada (p<0,006). A medida que decrece la circunferencia del cuello mejoran los valores de la presión de los universitarios (p<0,001). También, se observó que los universitarios con valores de glucemia de ayuno plasmática (p=0,003) y triglicéridos (p<0,001) elevados presentaron mayores valores de circunferencia del cuello. CONCLUSIÓN: la circunferencia del cuello se mostró un posible indicador de predicción para la detección del síndrome metabólico y sus componentes, en universitarios. .

Functional fusion expression of sunflower multicystatin in E. coli and its comparison with a single domain cystatin.

Identification of the molecular structure and novel biophysiological functions of plant cystatins or phytocystatins is of great interest in the field of molecular biology. The important requirements for these are the efficient production, purification and correctly folded forms of these proteins. We report here the cloning, easy expression and characterization of a sunflower multicystatin (SMC) as a functional fusion protein in E. coli. For the first time, the amplified cystatin coding region was expressed as a part of maltose-binding fusion protein using pMALc2X over-expression vector in TB1 strain of E. coli without affecting the recombinant bacterial growth. In comparison to the previously prepared recombinant SMC (rSMC), a high amount (~44 mg/L of bacterial cell culture) of purified fused SMC (fSMC) was obtained using single-step purification method. fSMC strongly inhibited papain activity in vitro as compared to Celosia single-domain cystatin. Purified fSMC may be used for basic biochemical, pharmacological or clinical studies without the cleavage of its fusion parts.

Cistatina C sérica: uma alternativa prática para avaliação de função renal? / Serum cystatin C: a practical alternative for renal function evaluation?

A taxa de filtração glomerular é o principal indicador de função renal em indivíduos saudáveis e doentes. Apesar de todo o desenvolvimento da medicina em nossos dias, ainda há dificuldade para definir-se essa taxa com precisão na prática diária. Marcadores precoces de lesão renal são importantes, porque a taxa de filtração glomerular se reduz antes do aparecimento dos sintomas ou sinais de insuficiência renal. A cistatina C tem sido apontada como uma alternativa, mas ainda não foi testada em muitas condições. Vantagens e desvantagens desse marcador foram aqui discutidas. Embora a determinação sérica da cistatina C comece a ser usada na prática clínica em todo o mundo, ainda não foram completamente esclarecidas suas limitações ou as situações em que está de fato indicada sua aplicação; por outro lado, a creatinina sérica (e sua depuração) é um marcador laboratorial facilmente acessível, de baixo custo, cujas limitações são bem conhecidas, que pode ser usado de forma rotineira para avaliação de função renal.

Glomerular filtration rate is the main marker of renal function in healthy in>dividuals and patients. Despite incontestable advances in medicine, it is still difficult to define precisely this test in clinical practice. Early markers of renal lesion are important, because glomerular filtration rate usually decreases before the first chronic renal failure symptoms or signs appear. Cystatin C has been pointed as an alternative, but it was not tested in many diseases. Advantages and disadvantages of this marker are discussed. Although serum cystatin C determination is increasingly being used in clinical practice worldwide, its limitations as well as the conditions its use is in fact indicated are not adequately established; on the other hand serum creatinine (and creatinine clearance) is an easily available and low cost laboratory marker with well-known limitations that can be used routinely in the assessment of renal function.

Parasitic Helminth Cystatin Inhibits DSS-Induced Intestinal Inflammation Via IL-10+F4/80+ Macrophage Recruitment

Many immune down-regulatory molecules have been isolated from parasites, including cystatin (cystain protease inhibitor). In a previous study, we isolated and characterized Type I cystatin (CsStefin-1) of the liver fluke, Clonorchis sinensis. To investigate whether the CsStefin-1 might be a new host immune modulator, we induced intestinal inflammation in mice by dextran sodium sulfate (DSS) and treated them with recombinant CsStefin-1 (rCsStefin-1). The disease activity index (DAI) increased in DSS only-treated mice. In contrast, the DAI value was significantly reduced in rCsStefin-1-treated mice than DSS only-treated mice. In addition, the colon length of DSS only-treated mice was shorter than that of rCsStefin-1 treated mice. The secretion levels of IFN-gamma and TNF-alpha in the spleen and mesenteric lymph nodes (MLNs) were significantly increased by DSS treatment, but the level of TNF-alpha in MLNs was significantly decreased by rCsStefin-1 treatment. IL-10 production in both spleen and MLNs was significantly increased, and IL-10+F4/80+ macrophage cells were significantly increased in the spleen and MLNs of rCsStefin-1 treated mice after DSS treatment. In conclusion, rCsStefin-1 could reduce the intestinal inflammation occurring after DSS treatment, these effects might be related with recruitment of IL-10 secreting macrophages.

Avaliação da função renal em pacientes submetidos à colecistectomia ou correção de hérnia de hiato por via laparoscópia / Renal function evaluation in patients submitted to cholecystectomy or hiatoplasty via laparoscopic surgery

O pneumoperitônio (PP), utilizado durante laparoscopia, produz oligúria transitória e diminui o ritmo de filtração glomerular (RFG) e o fluxo sanguíneo renal (FSR). O diagnóstico da disfunção renal aguda é rotineiramente baseado na elevação sérica da creatinina (Cr) e/ou na detecção de oligúria. A cistatina C (Cis C) tem sido estudada como um novo marcador de função renal. O objetivo foi avaliar a função renal, por meio da estimativa do RFG baseada nas concentrações sérica de Cr ou Cis C, de pacientes submetidos à videolaparoscopia.foram estudados 41 pacientes submetidos à colecistectomia ou à hiatoplastia pela via laparoscópica. A pressão intra-abdominal (PIA) foi mantida em 15 mm Hg durante a cirurgia. Amostras sanguíneas foram coletadas para mensuração dos valores séricos de vasopressina, Cr e Cis C antes da anestesia (M1), 30 min após a insuflação do PP (M2) e 30 min após a deflação do PP (M3). Quando a Cr foi utilizada para a estimativa do RFG, esta foi calculada pela fórmula de Cockcroft-Gault (RFG-CG). Quando a Cis C foi utilizada para o mesmo fim, a fórmula empregada foi a de Larsson (RFG-Larsson).os valores de Cis C aumentaram durante o estudo (M1 = M2 < M3; p < 0.05), enquanto os valores de Cr diminuíram nos momentos estudados, provavelmente decorrente da hemodiluição resultante da reposição volêmica durante o procedimento (M1 = M2 > M3; p < 0.05). Consequentemente, o RFG-Larsson (mL.min-1) diminuiu (M1 = 134,5 ± 38,2; M2 = 128,5 ± 33,8; M3 = 121,3 ± 33,7; M1 = M2 > M3) e o RFGCG aumentou durante os momentos estudados (M1 = 132,9 ± 37,9; M2 = 140,7 ± 45,4; M3 = 155,8 ± 57,0; M1 = M2 < M3).

Pneumoperitoneum (PP) used during laparoscopic procedure has been shown to produce transient oliguria and reduced glomerular filtration rate (GFR) and renal blood flow (RBF). The diagnostic of acute kidney injury is usually based on either an elevation of serum creatinine (Cr) or the detection of oliguria. A relatively new marker for detecting renal injury is the cystatin C (Cys C). Our goal was to evaluate the renal function through analysis of GFR estimated by concentration of serum Cys C and serum Cr during laparoscopic surgery.we evaluated 41 patients subjected to colecistectomy or hiatoplasty by laparoscopic approach. Intraperitonial pressure during PP was maintained in 15 mm Hg. Blood samples were collected for vasopressin, Cys C, and Cr measurements (before intubation (M1), 30 min after PP (M2), and 30 min after the deflation of PP (M3)). To estimate GFR we used Larsson formula to evaluate Cys C (GFR-Larsson) and Cockcroft-Gault formula to evaluate Cr (GFR-CG).the values of Cys C increased during the study (M1 = M2 < M3; p < 0.05). Cr values decreased during the study probably because the hemodilution effect caused by fluid replacement (M1 = M2 > M3; p < 0.05). Consequently, the GFR-Larsson (ml.min-1) decreased (M1 = 134.5 ± 38.2; M2 = 128.5 ± 33.8; M3 = 121.3 ± 33.7 with M1 = M2 > M3), while GFR-CG increased during the study (M1 = 132.9 ± 37.9; M2 = 140.7 ± 45.4; M3 = 155.8 ± 57.0 with M1 = M2 < M3). Person’s analysis showed better correlation between Cys C values and GFRLarsson (M1 = -0.96; M2 = -0.95; M3 = -0.94) versus Cr values and GFR-CG (M1 = - 0.65; M2 = -0.67; M3 = -0.78).

Cystatin C: a marker of renal function in transplant patients: [review] / Cistatina C: um marcador de função renal em pacientes transplantados: [revisão]

Assessment of renal function in patients submitted to kidney transplantation is of great importance in clinical practice, and the glomerular filtration rate is used as an indicator for this purpose. Measurement of serum creatinine is the most widely used method for the estimation of gromerular filtration rate. However, the disadvantages of this method are physiological and analytical influences (e.g., muscle mass, gender, certain antibiotics, bilirrubin, ketones) with the assay and inadequate sensitivity for the early detection of small declines in gromerular filtration rate. Cystatin C is a nonglycosylated low molecular weight (13 kDa) protein of the superfamily of cysteine proteinase inhibitors, which isconstantly produced by all nucleated cells. Due to its low molecular weight, cystatin C is freely filtered by the renal glomeruli and then almost completely reabsorbed and metabolized in the proximal tubules without interference from other low molecular weight proteins, thus permitting its use as a good marker of glomerular filtration. We present here a review of studies published so far regarding the use of cystatin C as a promising marker for the assessment of glomerular filtration in renal transplant patients.

A avaliação da função renal em pacientes submetidos a transplante renal é de grande importância na prática clínica, sendo utilizada para esse fim como indicador a medida da taxa de filtração glomerular. A determinação da creatinina sérica é o procedimento mais utilizado para essa avaliação, porém as desvantagens desse método são as influências fisiológicas e analíticas, por exemplo, massa muscular, sexo, certos antibióticos, bilirrubinas, cetonas, com o ensaio e inadequada sensibilidade para detecção precoce de pequenos declínios na taxa de filtração glomerular. A cistatina C é uma proteína não glicosilada de baixo peso molecular (13KDa), produzida constantemente em todas as células nucleadas, pertencente à superfamília das proteínas inibidoras da cisteína proteinase. É livremente filtrada pelos glomérulos renais devido ao seu baixo peso molecular, sendo a seguir quase totalmente reabsorvida e metabolizada nos túbulos proximais, não sofrendo influência de outras proteínas de baixo peso molecular, o que permite sua utilização como um bom marcador da filtração glomerular. Esta revisão aborda o uso da cistatina C como marcador promissor de avaliação da filtração glomerular em pacientes transplantados renais, com base em estudos até o momento realizados nesta população.

Partial Purification and Characterization of a Cysteine Protease Inhibitor from the Plerocercoid of Spirometra erinacei

Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargana and characterized its biochemical properties. The cysteine protease inhibitor was purified by sequential chromatographies using Resource Q anion exchanger and Superdex 200 HR gel filtration from crude extracts of spargana. The molecular weight of the purified protein was estimated to be about 11 kD on SDS-PAGE. It was able to inhibit papain and 27 kDa cysteine protease of spargana with the ratio of 25.7% and 49.1%, respectively, while did not inhibit chymotrypsin. This finding suggests that the cysteine protease inhibitor of spargana may be involved in regulation of endogenous cysteine proteases of the parasite, rather than interact with cysteine proteases from their hosts.

Predictive value of serum neutrophil gelatinase-associated lipocalin and cystatin C as markers for contrast nephropathy in patients undergoing coronary angiography

Contrast induced-nephropathy is a more frequent and potentially serious complication after coronary angiography. Very few biomarkers exist for monitoring contrast induced-nephropathy. Recently, serum neutrophil gelatinase-associated lipocalin [NGAL] represents a novel biomarker for early identification of acute kidney injury. The present work aimed to test the hypothesis that NGAL could represent an early biomarker for kidney injury and to assess the relation ship between NGAL and serum creatinine and cystatin C, in patients with normal serum creatinine undergoing percutaneous coronary angiography. The study was performed on thirty non-diabetic patients with normal serum creatinine, undergoing coronary angiography due to corollary artery disease. All patients were mateched for age and body mass index. following full clinical examination, fasting blood sample were withdrawn for estimation of blood glucose. glycosylated hemoglobin, lipid profile. creatinine as well as NGAL and cystatin C before coronary angiography. Another blood samples were taken 4 and 24 hours after coronary angiography for all patients for evaluation of serum creatinine, NUGL and cystatin C. There was a significant increase in serum NGAL level 4 hours and 24 hours after coronary interventions when compared to the baseline value before coronary- angiography Before coronary angiography, serum NGAL was positively correlated with serum creatinine, and cystatin C. in multiple regression analysis, serum creatinine was the only predictor of serum NGAL. Serum NGAL, 4 hour after coronary angiography, correlated with serum ereatinine only in simple and multiple regression analysis. On the other hand, serum cystatin C level increased significantly only 24 hour after coronary angiography compared to the baseline value before coronary angiography. In a simple regression analysis serum cystain C correlated positively to systolic and diastolic blood pressure, Serum creatinine and serum NGAL, before Coronary angiography. In multiple regression analysis, serum creatinine and systolic blood pressure were the predictors of serum cystatin C. Serum NGAL and cystatin C could be valuable in the detection of acute renal impairment after coronary angiography. However current diagnostic methods such as. Serum creatinine or cystatin C measurements only respond after renal function has deteriorated. Therefore, the presence of new early markers for renal injury such as NGAL, can initiate proper management of acute renal failure within hours rather than days of the insult

Prokaryotic expression of OC-IdeltaD86 (Oryzacystatin-IdeltaD86) gene and analysis of its activity / 生物工程学报

According to the amino acids sequence of OC-IdeltaD86 gene and Escherichia coli codon usage, we synthesized this gene by overlap extension PCR method with 7 oligonucleotides DNA fragments. The PCR fragment was inserted into pGEM-T-easy vector and the recombined plasmid was named pGEM-T-OC-IdeltaD86. Two oligonucleotides into which the BamH I and Xho I sites were introduced were designed and synthesized based on pGEM-T-OC-IdeltaD86 and pet21b, and the PCR fragment into which the BamH I and Xho I sites were introduced was obtained. After digesting it with BamH I and Xho I, OC-IdeltaD86 gene was cloned into the corresponding sites of pet21b and obtained prokaryotic expression vector pet21b-OC-IdeltaD86. OC-IdeltaD86 gene was expressed in E. coli (BL21(DE3)plysS) after IPTG(Isopropyl beta-D-1-thiogalactopyranoside) inducement for 5 hours. The fusion protein of OC-IdeltaD86:6His gene accounted for 11.4% of total protein and 16.4% of soluble protein, which had been successfully purified by Ni-NTA and concentrated by PEG20000. This protein can effectively inhibit papain activity in vitro and may be used in anti-nematode research in vivo.

Cloning of Rcet3, a novel gene related to family 2 cystatins / 南方医科大学学报

<p><b>OBJECTIVE</b>To clone the full-length Rcet3 gene, a novel gene related to family 2 cystatins, from mouse testis or other tissues.</p><p><b>METHODS</b>Rcet3 gene was cloned using digital differential display (DDD) and RT-PCR was performed for cloning the full-length Rcet3 gene from adult mouse testis cDNA library with sequence analysis.</p><p><b>RESULTS</b>Rcet3 cDNA was 610 bp in length, consisting of 4 exons to encode a protein with 140 amino acid residues. The encoded protein contained a potential signal peptide and a cystatin domain, but lacked critical consensus site important for cysteine protease inhibition. These characteristics could be seen in the Cres subgroup related to the family 2 cystatins. Rcet3 was specifically expressed in adult mouse testis, epididymis and the cerebrum, but at higher levels in the testis than in the epididymis and cerebrum.</p><p><b>CONCLUSION</b>Rcet3 may be a new member of Cres subgroup of family 2 cystatins.</p>

Avaliação da função e da lesão renal: um desafio laboratorial: [revisão] / Evaluation of renal function and damage: a laboratorial challenge: [review]

Atualmente a doença renal é um grande problema de saúde pública, que acomete milhares de pessoas no Brasil e no mundo. O estudo da função e dos diversos processos patológicos renais tem despertado o interesse de muitos pesquisadores, principalmente no campo do desenvolvimento de testes que auxiliem os médicos a estabelecer um diagnóstico precoce, classificar a doença de base, obter prognóstico seguro e monitorar terapêutica medicamentosa. Neste artigo sete marcadores de função e de lesão renal são avaliados: uréia, creatinina, cistatina C, proteinúria, dismorfismo eritrocitário, microalbuminúria e fração hepática das proteínas ligadas a ácidos graxos. É apresentado um breve histórico da utilização clínica e da fisiopatologia de cada um deles, seguidas de sua aplicabilidade e dos avanços técnicos e metodológicos disponíveis. Apesar de melhorias terem sido conseguidas e incorporadas à prática laboratorial, nenhum marcador atualmente disponível é completamente eficaz em analisar a função e/ou a lesão renal de forma precisa, sendo imprescindível o conhecimento de todos eles para uma correta avaliação desses testes comuns na rotina laboratorial.

Nowadays, renal disease is an important public health problem, affecting millions of people in Brazil and in the world. The study of renal function and renal pathologic processes has aroused the interest of researchers, mainly in the field of development of new assays that could aid physicians in establishing early diagnosis, better classifying the disease, obtaining better outcome and monitoring drug therapeutics. In this article, seven laboratory markers of renal function or damage are evaluated: urea, creatinine, cystatin C, proteinuria, dysmorphic erythrocytes, microalbuminuria and liver-type fatty acid binding protein (L-FABP). For each one of them, a short historical report of its clinical utility and physiopathology is presented. Then technical and methodological approaches are described as well as its utility in clinical management of kidney patients. Although improvements have been reached and incorporated in laboratorial practice, none of these markers is effective enough to define precisely kidney function and/or damage and an extensive understanding of all of these markers is crucial to correct evaluate renal function.

Avaliação do ritmo de filtração glomerular / Assessment of glomerular filtration rate

A medida do ritmo de filtração glomerular (RFG) é a prova laboratorial mais utilizada na avaliação da função renal. Para tanto, usam-se marcadores indiretos, como as determinações de creatinina e cistatina C no sangue, ou procede-se à determinação do RFG propriamente dito, com indicadores como inulina; contrastes iodados, marcados ou não; e outras substâncias. O exame mais solicitado para avaliação do RFG no laboratório de patologia clínica é a dosagem da creatinina sérica. Em algumas condições, entretanto, o resultado encontrado da creatinina sérica deve ser corrigido (através da utilização de fórmulas que levam em consideração características próprias do indivíduo) para ser devidamente interpretado. De fato, a inulina ainda é vista como marcador ideal de filtração glomerular, mas seu uso não se destina à prática clínica, de modo que ainda hoje persiste a busca por testes adequados para uso rotineiro.

Glomerular filtration rate (GFR) determination is the most frequently used laboratorial test to evaluate renal function. Indirect markers as blood determination of creatinine and cystatin C are used with this purpose, as well as the direct determination of GFR, with indicators like inulin; iodated contrasts, radioactive or not; and others. Serum creatinine is the test that is most commonly performed in order to evaluate GFR in the clinical pathology laboratory. However, in some conditions, aiming at the adequate interpretation of the test, the result of serum creatinine must be corrected (by using formulas that include individual characteristics of the subjects). In fact, inulin is still seen as the ideal marker of glomerular filtration, but its use is not directed to clinical practice; then the search for appropriate tests for routine use continues.

Avaliação da filtração glomerular através da medida da cistatina C sérica: [revisão] / Glomerular filtration evaluation employing serum cystatin C measurement: [review]

A avaliação acurada da função renal através da medida da filtração glomerular (FG) é fundamental na rotina clínica, pois é parte decisiva do diagnóstico e terapêutica. A dosagem sérica de creatinina é o método mais usado, embora apresente limitações, como interferências na dosagem e baixa sensibilidade na detecção de graus menos avançados de perda de função renal. Outros métodos, como depuração de inulina, iohexol, 125I-iotalamato e 51Cr-EDTA, também são descritos com o mesmo propósito, mas são complexos e caros. Desta forma, investigadores ainda buscam um marcador ideal para analisar a função renal. Neste contexto, se encaixam os estudos com a cistatina C, uma substância endógena, que vem sendo relatada como um indicador confiável e de fácil execução. A cistatina C é um membro da família dos inibidores da cisteína protease e está presente em uma variedade de células nucleadas,sendo produzida de forma constante. A sua medida, comparada à da creatinina, sofre menos interferências e apresenta maior acurácia na detecção dereduções incipientes da função renal. O objetivo desta revisão é descrever a medida da cistatina C como uma alternativa confiável para avaliar a FG,analisando valores de referência e usos clínicos.

An accurate evaluation of renal function with glomerular filtration rate (GFR) measurement is essential in clinical practice, as it defines diagnostic andtherapeutic decisions. Serum creatinine is the most utilized method. However, it has several limitations, such as the influence of drugs and endogenoussubstances on its measurement, and a low sensitivity for detecting less advanced degrees of renal dysfunction. Other well-known methods, such as theclearances of insulin, iohexol, 125I-iothalamate, and 51Cr-EDTA have also been employed with the same purpose; however, these methods can be complexand expensive. Therefore, investigators are still searching for simpler markers to analyze renal function. Cystatin C, an endogenous substance, has beendescribed as a reliable and easy-to-perform indicator of GFR. Cystatin C is a member of the family of cystein protease inhibitors and is constantly produced by nucleated cells. Its measurement, when compared to creatinine, suffers less interference and is more accurate for the detection of incipient decreases of renal function. The aim of this review is to analyze the details of cystatin C measurement, such as reference values and clinical application.

Assessment of the Accuracy and Precision of Cystatin C-based GFR Estimates and Cr-based GFR Estimates in Comparison with Cr51-EDTA GFR / 대한진단검사의학회지

BACKGROUND: Cystatin C (cysC) is said to be an ideal marker for glomerular filtration rate (GFR), independent of external factors such as age, nutrition and inflammation. The authors compared the accuracy and precision of cysC-based and creatinine (Cr)-based GFR estimates using Cr51-EDTA GFR method as a reference. METHODS: Serum concentrations of cysC and Cr were measured in adults over 17 yr (n=170) and children below 17 yr (n=79) who had had GFR estimated by Cr51-EDTA method. CysC-based GFR was estimated by the formula of Thierry [CysC-based GFR estimates (mL/min/1.73 m2)=78 x (1/cysC, in mg/L)+4] and Cr-based GFR by the formula of modified Modification of Diet in Renal Disease [MDRD II, Cr-based GFR estimates (mL/min/1.73 m2)=186 x (Scr)(-1.154) x (Age)(-0.203) x 0.742 (for a female patient) x 1.212 (for a black patient). RESULTS: In comparison with Cr51-EDTA GFR, in children below 17 yr, the bias +/- standard deviation (SD) of cysC-based and Cr-based GFR estimates were 7.5 +/- 6.1 and 106.5 +/- 98.2, respectively, in the range of below 90 of Cr51-EDTA GFR (mL/min/1.73 m2), and 33.7 +/- 33.0 and 174.4 +/- 18.8 in the range of over 90. In adults over 17 yr, the respective figures were 13.1 +/- 11.0 and 17.4 +/- 29.8 in below 90, and 21.2 +/- 20.1 and 83.6 +/- 108.8 in over 90 of Cr51-EDTA GFR. CONCLUSIONS: CysC-based GFR estimates show acceptable ranges of biases over the whole age and GFR ranges. CysC-based GFR estimates is considered to be the marker for GFR, which could be used without limitation of age and GFR ranges.

Evaluation of serum cystatin C as a marker for detection of early renal function impairment in children with chronic liver disease

Evaluation of renal failure is essential in patients with decompensated liver cirrhosis, because a significant proportion of them manifest reduced glomerular filtration rate [GFR]. Careful assessment of GFR is critically important for prognosis because the indicators of renal function are sensitive markers of severity of liver dysfunction in cirrhosis and are better predictors of patient survival than estimating hepatic dysfunction. Plasma creatinine concentrations and calculated creatinine clearance are of limited values as GFR markers in patients with chronic liver diseases [CLD] especially liver cirrhosis. Recently, assessment of serum cystatin C concentrations was proposed as a possible indicator of early GFR changes in such patients. The aim of this work was to study the utility of measurement of serum cystatin C level as a marker of early detection of renal impairment in patients with CLD and to assess its correlation to the severity of liver dysfunction. This study was conducted on 30 children [17 males and 13 females] with CLD. Their ages ranged from 2 to 16 years. Twenty healthy children with matched age and sex were chosen as a control group. They were selected from those admitted to the Hepatology Unit of Pediatric Department, Tanta University Hospitals. In this study all patients were subjected to the following: full clinical history, through physical examination, abdominal ultrasonography, histopathological assessment of liver biopsy and laboratory investigations. The latter included complete blood count, liver function tests, complete urine analysis, blood urea, serum creatinine, creatinine clearance [CrCI], hepatitis markers as well as measurement of serum cystatin concentrations by particle induced immunonephelometry. Control children were subjected to the whole previous investigations except liver biopsy. Severity of liver dysfunction in studied patients was classified into grades A, B and C according to modified Child-Pugh' classification. This study showed that mean CrCl values were significantly reduced in patients [77.03 +/- 17.4 ml/min/1.73m[2]] compared to controls [86.7 +/- 9.2 ml/min/1.73m[2]]. Mean CrCI values were impaired in 3 [10%] patients. All of them had ascites. Serum cystatin C levels were significantly higher in the studied patients [1.02 +/- 0.55mg/L] compared to controls [0.38 +/- 0.10mg/L], and significantly higher in grade C patients [1.35 +/- 0.65mg/L] than in those with grades B [0.92 +/- 0.46 mg/L] and A [0.73 +/- 0.29 mg/L]. Serum cystatin C levels were high in 10 [30%] patients of whom 70% [7/10] had ascites. Regarding ascitic patients, there was a significant reduction in CrCl values in ascitic compared to non-ascitic patients. Furthermore, there was a significant increase in serum cystatin C levels in ascitic compared to non-ascitic patients. Serum cystatin C correlated significantly with CrCI and severity of CLD as assessed by its correlation with liver function tests and Child's scores. ROC curve plots demonstrated that the area under the curve [AUC] of cystatin C [0.92] was greater than that of CrCI [0.76] and serum creatinine [0.58]. Therefore, sensitivity, specificity and diagnostic accuracy of cystatin C were higher than those of CrCl and both were better than those of serum creatinine. The positive and negative predictive values of serum cystatin C were higher than those of CrCl and serum creatinine. From this study, we can conclude that serum cystatin C is more accurate and simple than serum creatinine and creatinine clearance as a marker of GFR changes for prediction of early renal impairment in patients with CLD. Furthermore, serum cystatin C concentration was significantly high in patients with CLD that was correlated with the severity of liver dysfunction. Measurement of serum cystatin C may be recommended or at least added to serum creatinine in the routine assessment of early GFR changes in patients with CLD. However, further prospective comparative studies on a large scale with a gold standard method for measuring GFR are required to evaluate serum cystatin C concentration in different stages of renal impairment In children with CLD

Cystatine C biochemical marker for renal dysfunction in coronary artery bypass randomized double blinded comparison between the effects of sevoflurane and isoflurane

Volatile anesthetics exert significant protection against myocardial ischemia and excitotoxic cardiomyocyte death. One of the mechanisms by which volatile anesthetics induce protection in myocytes is pharmacological preconditioning, the activation of a potent endogenous protective mechanism in cardiac tissue against a variety of important stressors. Laboratory investigations further stress the concept that volatile anesthetics may protect endothelial and smooth muscle cells, implying that anesthetic protection might beneficially affect a much wider variety of tissues including the brain, spinal cord, liver, and kidneys. After written informed consents were obtained from all patients, 40 patients scheduled for elective CABG, they were divided into 2 groups, 20 patients each group, group I [isoflurane group], group II [sevoflurane group]. All patients received midazolam for premedication. Anesthesia was induced in all patients with propofol, fentanyl and the muscle relaxant vecuronium. Blood samples were obtained preoperatively, at arrival in the intensive care unit, and 24, 48, and 72h after surgery; they were stored at -20°C until analysis. Creatinine level and Cystatine C [CysC] assays were used as markers for renal dysfunction. Sevoflurane-treated patients required significantly more phenylephrine than did isoflurane-treated patients to maintain blood pressure above 50mmHg. However, there was no difference in mean arterial blood pressure between both groups. CysC concentrations significantly increased immediately postoperatively and peaked at 48 h after surgery for both groups [time effect, p<0.001]. Cystatine C concentrations were markedly higher for isoflurane-treated patients than for sevoflurane-treated patients. Plasma creatinine concentrations were slightly, but not significantly, increased in isoflurane-treated patients. The study concluded that, sevoflurane could be administered safely during CABG without affecting renal function. Because this study only evaluated immediate perioperativc effects of sevoflurane, we can only speculate on potential beneficial long-term effects of this treatment. Future studies should address this important issue