Isolation and characterization of an antithrombin III variant with reduced carbohydrate content and enhanced heparin binding

Two distinct forms of antithrombin III were isolated by chromatography of normal human plasma on heparin-Sepharose. The predominant antithrombin species present (AT-III alpha), which eluted from the affinity column in 1 M NaCl, was identified as the antithrombin III form which has been previously characterized. Ionic strength of the buffer was increased to elute a variant form of antithrombin III, designated as AT-III beta. The molecular weight of AT-III beta is less than that of AT-III alpha, but physicochemical studies do not indicate measureable differences in the polypeptide portion of the proteins. Carbohydrate determination revealed the sole detectable structural difference in the two antithrombins: levels of hexosamine, neutral sugars, and sialic acid in AT-III beta were all 25-30% less than in AT-III alpha. Kinetic studies of thrombin inactivation by both antithrombins, in the presence of nonsaturating amounts of heparin, indicated that AT-III beta inhibited thrombin more rapidly. AT-III beta is also distinguishable from AT-III alpha on the basis of heparin-binding affinity estimated from titration of protein fluorescence with heparin. Thus, antithrombin III exists as two molecular entities in human plasma which differ both structurally, in carbohydrate content, and functionally, in their heparin-binding behavior.     

Plasminogen activator in nephrotic syndrome

Plasminogen activators were studied in blood urine in 207 patients with nephrotic syndrome of different etiological forms. The blood plasminogen activator activity was decreased in chronic glomerulonephritis, SLE, systemic vasculities as result of great level of inhibitors (L2M), penetration of enzymes to abdominal and pleural transudates, excretion to urine. The blood plasminogen activator activity and urokinase level in chronic glomerulonephritis was dependent on the degree of nephrotic syndrome. The plasminogen activator in amyloidosis was sharply elevated because of permanent irritability of endothelial wall by amyloid mass. Venous occlusion caused the release of plasminogen activator to blood only in more favourable clinical course of nephrotic syndrome.     

Plasma and urinary lipids and lipoproteins during the development of nephrotic syndrome induced in the rat by puromycin aminonucleoside

This study was undertaken to ascertain whether the alterations of plasma lipoproteins found in nephrotic syndrome induced by puromycin aminonucleoside were due to nephrotic syndrome per se, or, at least in part, to the aminonucleoside. The purpose of the present study was to investigate the changes in plasma and urinary lipoproteins during the administration of puromycin aminonucleoside (20 mg/kg for 7 days) and the subsequent development of nephrotic syndrome. Since massive albuminuria occurred after 6 days of treatment, the time-course study was divided into two stages: pre-nephrotic stage (day 1–5) and nephrotic stage (day 6–11). In pre-nephrotic stage the plasma level of fatty acids, triacylglycerol and VLDL decreased while that of phospholipid, cholesteryl esters and HDL remained constant. Plasma apolipoprotein A-I tended to increase (40% increase at day 5). At the beginning of nephrotic stage (day 6) the concentration of plasma albumin dropped to a very low level, while that of apolipoprotein A-I increased abruptly (4-fold increase) and continued to rise, although less steeply, in the following days. The plasma concentration of HDL followed the same pattern. Plasma VLDL and LDL increased at a later stage (day 9). Plasma apolipoprotein A-I was found not only in HDL (1.063–1.210 g/ml) but also in the LDL density class (1.025–1.050 g/ml). In the pre-nephrotic stage lipoproteinuria was negligible, while in the early nephrotic stage the urinary loss of plasma lipoproteins consisted mainly of HDL. These observations indicate that puromycin aminonucleoside alters plasma lipoproteins by lowering VLDL and increasing HDL. It is likely that the early and striking increase of plasma HDL found in nephrotic rats is related to a direct effect of the drug on HDL metabolism.     

Dynamic tests and FSH biological activity in female rats with acute nephrotic syndrome

To investigate the pituitary-ovarian status during the acute state of the nephrotic syndrome, a sequence of experiments were undertaken in adult female rats after a single dose of the puromycin aminonucleoside (PAN). The functional condition of the hypophyseal-ovarian unit was determined in control and nephrotic rats by two dynamic tests. In the first one, 10 days after PAN or placebo administration female rats were stimulated with LHRH (300 ng/100 g body wt) and samples were collected at 0, 20, 40, 60 and 80 min after releasing factor administration. The second dynamic test, which was performed in control and nephrotic rats, consists of one (day 10 after PAN) or four (between days 7-10 after PAN) doses of hCG (8 UI), respectively. In all cases, serum samples were collected on day 10. In addition, the relative in vitro biological activity of FSH from control and nephrotic rats before and after LHRH stimulus was determined. The results reveal that after a stimulatory dose of LHRH the secretion of LH was significantly diminished in nephrotic rats at all registered times. By contrast, normal response was observed in terms of FSH secretion in nephrotic females. On the other hand, no ovarian response, in terms of progesterone or estradiol synthesis, was observed in nephrotic rats after either one or four stimuli with hCG Interestingly, in spite of the normal or high concentrations of FSH, the biological activity of FSH was totally abolished in nephrotic rats. On the whole, the results from this study indicate that the nephrotic syndrome had a harmful effect on the pituitary-ovarian unit, and strongly suggest that the endocrine dysfunction could be initiated at the hypophysial level; even though a specific ovarian damage is also predictable.     

Molecular dissection of laminin alpha 5 in vivo reveals separable domain-specific roles in embryonic development and kidney function

Laminins are a family of basement membrane proteins with diverse roles in fundamental developmental processes such as epiblast polarization and gastrulation, as well as in organ development and function. We have focused on the laminin alpha1 and alpha5 chains, the ancestral laminin alpha chains required for development. To elucidate the unique functions of laminin alpha1 and alpha5 and their COOH-terminal LG domains, we have produced a collection of laminin knockout and transgenic mice expressing full length and chimeric laminin alpha5/alpha1 chains. Crossing the transgenes onto the Lama5-/- background generates "pseudo-knockins", so called because endogenous laminin alpha5 is replaced by transgene-encoded proteins. Expression of a chimera with the entire alpha5LG domain replaced by alpha1LG had minimal ameliorative effects on the defects observed in Lama5-/- embryos. In contrast, high level expression of a chimera with only the alpha5LG3-5 tandem replaced by alpha1LG3-5 completely rescued defects in digit septation, neural tube closure, placental labyrinth morphology, lung lobe septation, hair growth, and vascularization of kidney glomeruli. These mice were viable for several months, but they developed a lethal nephrotic syndrome. Our results show that: (1) the laminin alpha5LG1-2 tandem plays an essential role during development and harbors the great majority of the functionality of the alpha5LG domain; and (2) the alpha5LG3-5 tandem serves as a novel determinant required for the kidney's glomerular filtration barrier to plasma protein.     

Salmon antithrombin has only three carbohydrate side chains, and shows functional similarities to human beta-antithrombin.

Antithrombin, a major coagulation inhibitor in mammals, has for the first time been cDNA cloned from a fish species. The predicted mature liver antithrombin of Atlantic salmon (Salmo salar) consists of 430 amino acids and shows about 67% sequence identity to mammalian and chicken antithrombins. Due to a single nucleotide replacement, Asn135 of the antithrombin in higher vertebrates is substituted by Asp in the salmon homolog. Hence, in contrast to the vertebrate antithrombins known so far, salmon antithrombin lacks the potential glycosylation site located close to the heparin binding site. The existence of only three N-linked side chains is evidenced by the sequential removal of three carbohydrate chains from salmon antithrombin during timed-digestion with N-glycosidase F. The high heparin binding affinity of the salmon inhibitor, Kd of 2.2 and 48 nM at I = 0.15 and 0.3, respectively, is very similar to that of the minor human isoform beta-antithrombin, which is not glycosylated at Asn135. Furthermore, the invariant third-position Ser137 at this glycosylation site of mammalian and chicken antithrombins is substituted by Thr in the salmon, a replacement that has been shown to induce full glycosylation in human antithrombin. Thus a rapidly reacting pool of antithrombin may have evolved in two different ways: absence of a glycosylation site in lower vertebrates vs. incomplete glycosylation of a part of the circulating antithrombin in higher vertebrates. Salmon antithrombin appears to have three complex oligosaccharide side chains containing sialic acid terminally linked alpha(2-3) to galactose, while trace amounts of Galbeta(1-4)GlcNAc suggest microheterogeneity due to partial loss of sialic acid.     

Renal leptin in experimental nephrotic syndrome

Leptin is a fat derived hormone involved in the regulation of metabolism and body composition. The kidney is the principle organ responsible for elimination of circulating leptin. Our aim is to evaluate if the nephrotic kidneys participate in the metabolism of leptin by comparing the serum leptin level in renal veins and in their renal arteries and to study the relationship between leptin and lipoprotein levels in healthy and nephrotic rats. Methods: Rats were divided into two equal groups: group 1 in which experimental nephrotic syndrome was produced by injecting them intraperitoneally with a supernatant of the homogenized mixture of their own kidney (obtained by previous unilateral nephrectomy) and complete Freund’s adjuvant. Another group constituted the control group. Leptin and lipid profile were estimated in blood samples of renal veins and renal arteries. There was a highly significant increase in leptin and lipid profile levels in the nephrotic rats compared with the normal group. There was a high significant decrease in leptin in the renal venous blood compared with its level in the renal arterial blood of normal and nephrotic rats. This work has stressed the involvement of kidney and the nephrotic renal tissue in the process of leptin metabolism and lipogenesis.     

In vivo platelet activity and serum albumin concentration in nephrotic syndrome

To clarify the relationship between serum albumin concentration and in vivo platelet activity in nephrotic syndrome, Beta-thromboglobulin (B-TG) levels and circulating platelet aggregation ratio (PAR) were determined in 25 nephrotic patients. PAR levels were significantly decreased compared with the controls and showed a positive correlation with serum albumin concentrations. The values of B-TG were high in all nephrotic patients and showed an inverse relationship with serum albumin levels. In addition, increased B-TG levels correlated with decreased PAR values. Therefore serum albumin plays a regulatory role in the activity of circulating platelets in the nephrotic syndrome. Prospective longitudinal studies must be done to elucidate the usefulness of platelet inhibitors as a prophylactic therapy of intravascular thrombosis in nephrotic patients.     

原发性肾病综合征患儿家长对疾病营养知识的调查与分析

目的:研究原发性肾病综合征患儿家长疾病营养知识,分析其相关影响因素。方法:选取2014年5月到2015年5月在我院门诊及住院的原发性肾病综合征患儿150例,应用自制问卷对患儿家长的一般资料及营养知识进行调查,应用非条件Logistic回归分析患儿家长营养知识影响因素。结果:150例原发性肾病综合征患儿家长对基本营养知识回答的正确率介于30.7%-96.7%,对具体营养知识回答的正确率为2.0%-42.0%;Logistic回归分析显示:家长文化程度、患儿病程、居住地、发病情况以及首次就诊是否接受营养指导均是家长疾病营养知识影响因素。结论:原发性肾病综合征患儿家长对疾病具体营养知识知晓率低,应根据影响因素对家长进行营养宣教。     

Serum malondialdehyde levels,myeloperoxidase and catalase activities in patients with nephrotic syndrome

Abstract

Objectives

Some studies have indicated the pathophysiological importance of reactive oxygen species (ROS) in patients with nephrotic syndrome. Myeloperoxidase (MPO) is a leukocyte-derived enzyme-generating ROS that has been proposed to exert a wide array of pro-atherogenic effects throughout all stages of the atherosclerotic process. The aim of this study was to investigate the serum malondialdehyde (MDA) levels, MPO and catalase activities in patients with adult nephrotic syndrome.

Patients and Methods

Twenty-four patients with nephrotic syndrome and 24 healthy controls were enrolled. Serum MPO activity, catalase activity, and MDA levels were assessed.

Results

Serum MPO activity and MDA levels were signi?cantly higher in patients with nephrotic syndrome than controls (both, P < 0.001), while catalase activity was signi?cantly lower (P < 0.001). Serum catalase activity was found to be significantly correlated with MPO activity (r = ?0.417, P = 0.003) and MDA levels (r = ?0.532, P = 0.007). The serum MDA levels were also found to be significantly correlated with MPO activity (r = 0.419, P = 0.003).

Conclusions

We concluded that serum MPO activity and oxidative stress were increased and that serum catalase activity was decreased in patients with adult nephrotic syndrome. In addition, these results indicate that increased MPO activity is associated with an oxidant–antioxidant imbalance that may contribute to atherosclerosis in patients with adult nephrotic syndrome.     

成人原发肾病综合征患者血尿酸与血脂代谢的关系

目的:研究成人原发肾病综合征(PNS)患者高尿酸血症的患病率及其与血脂代谢的关系。方法:将109例成人PNS患者根据其尿酸水平分为高尿酸血症组与血尿酸正常组,检测患者的血脂、血清脂蛋白、ALB及24小时尿蛋白定量,分析成人PNS患者高尿酸血症的患病率,比较高尿酸血症组与血尿酸正常组PNS患者的一般情况及血脂水平,并分析PNS患者血尿酸水平的相关因素。结果:成人PNS患者高尿酸血症的发病率为24.77%;高尿酸血症组患者TG及24小时尿蛋白定量水平明显高于血尿酸正常组(P0.01),两组患者TC、LDL-C、HDL-C、Apo AI及Apo B比较差异无统计学意义(P0.05);成人PNS患者血尿酸水平与TG及24小时尿蛋白定量水平呈正相关(r=0.350,P=0.001;r=0.533,P=0.014),与TC、LDL-C、HDL-C及ALB无明显相关性(P0.05)。结论:成人PNS患者高尿酸血症的发生率较高,高尿酸血症患者具有更高的TG及尿蛋白水平,且成人PNS患者血尿酸水平与TG及24小时尿蛋白定量具有相关性,应高度重视成人PNS患者的血尿酸水平。     

Studies on urinary gamma-glutamyl transpeptidase in nephrotic syndrome patients

(1) Variations in the levels of GGT were measured in urine specimens taken in the early morning in control and in 20 consecutive adult patients with uncomplicated nephrotic syndrome. (2) The urinary GGT activity was increased in all cases of nephrotic syndrome patients investigated with different etiology. (3) A significant correlation was found between urinary GGT activity and serum albumin (r = 0.727) but not with serum cholesterol (r = 0.129). (4) These findings suggest that enhanced excretion of urinary GGT may be stimulated by decreased albumin concentration or oncotic pressure but does not appear to be due to leakage from plasma. (5) A systematic study on urinary GGT showed that GGT activity was decreased to the upper limit of normal control values in nephrotic syndrome patients after remission.     

Increased glomerular atrial natriuretic peptide receptor affinity in experimental nephrotic syndrome.

Atrial natriuretic peptide (ANP) is a cardiac hormone with natriuretic and diuretic effects. To better define the ANP hormonal system in the nephrotic syndrome, a condition associated with renal sodium retention, we undertook a study of glomerular ANP receptors in rats with puromycin aminonucleoside-induced nephrotic syndrome and in pair-fed controls. Nephrotic rats had significantly decreased serum albumin and total protein and significantly increased serum cholesterol, triglycerides and 24 hour urinary protein excretion. Plasma level of atrial natriuretic peptide was similar in both groups of rats. Competition binding inhibition studies in isolated glomeruli demonstrated one binding site in both groups of rats. The density of ANP binding sites in isolated glomeruli was similar in nephrotic and pair-fed rats while the binding affinity was increased significantly in the nephrotic rats. This is the first study to demonstrate alterations in renal ANP receptors in the nephrotic syndrome. Further studies will be necessary to determine whether alterations in glomerular ANP receptors contribute to renal sodium retention in the nephrotic syndrome.     

Effect of human erythropoietin (hEPO) treatment on anemia in ICR-derived glomerulonephritis (ICGN) mice.

ICR-derived glomerulonephritis (ICGN) mice are a novel inbred strain with hereditary nephrotic syndrome and are thus considered a good animal model of human idiopathic nephrotic syndrome. In the present study, we investigated the effect to erythrocyte production by human erythropoietin (hEPO) treatment in ICGN mice during the early nephrotic stage. Erythrocyte count, hemoglobin concentration and hematocrit value in hEPO-treated (5 U/body/day, for 5 days) ICGN mice were recovered to the levels found in normal ICR mice. In addition, there was no correlation between plasma creatinine level, a marker of renal function, and erythrocyte count after hEPO treatment. Therefore, anemia in ICGN mice may be caused by decreased production of EPO in the kidney following progressive parenchymal damage.     

Characterization of glycosaminoglycans in urine from patients with nephrotic syndrome and control subjects, and their effects on lipoprotein in lipase

Previously we found that the α₁-acid glycoprotein fraction from urine of patients with the nephrotic syndrome stimulated the lipoprotein lipase reaction in vivo and in vitro. The activator was separated from the α₁-acid glycoprotein and identified as a glycosaminoglycan. The studies reported here were undertaken to characterize and quantify the glycosaminoglycans contained in urine of patients with the nephrotic syndrome and to compare these to the glycosaminoglycans in urine of control subjects. We found that free low molecular weight glycosaminoglycans, heparan sulfate and chondroitin 4-sulfate, are excreted in both patients with the nephrotic syndrome and controls, however, patients with the nephrotic syndrome excreted much less of both glycosaminoglycans. The free form of heparan sulfate was found to be the activator which stimulated the lipoprotein lipase reaction in vitro in the presence of apolipoprotein C_II. In addition, the urine from patients with the nephrotic syndrome contained a protein-glycosaminoglycan complex which was absent in control urine. Glycosaminoglycans in the complex could be released by papain digestion or by trichloroacetic acid. Our evidence indicates that this glycosaminoglycan fraction is a low charge form of chondroitin sulfate.     

Urinary excretion of LATS in a patient with Graves' disease complicated by nephrotic syndrome

A case report of a female patient with Graves' disease complicated by nephrotic syndrome with high LATS activity in urinary gamma-globulin is presented. When in the hyperthyroid state with high LATS activity in the serum, she was treated with antithyroid drugs, excess iodine, and finally radioisotopes. Mild hypothyroidism occurred transiently without any significant change in serum LATS activity. Nephrotic syndrome suddenly appeared. Urinary IgG was purified by salting out with ammonium sulfate, DEAE and protein A-Sepharose, and LATS activity in the purified urinary IgG fraction was demonstrated. The specific activity of LATS activity in urinary IgG protein was slightly lower than that of the serum. This case is the first demonstration of LATS activity in urine from a patient with hyperthyroidism and nephrotic syndrome.     

原发性及狼疮性肾病综合征患者血清PAI-1、Lp(a)水平变化及其临床价值探讨

目的:探讨原发性及狼疮性肾病综合征患者纤溶酶原激活物抑制因子1(PAI-1)和血清脂蛋白a[Lp(a)]的水平变化及其检测的临床应用价值。方法:选取病理类型明确,临床初诊为肾病综合征的患者138例。其中原发性肾病综合征70例,为PNS组;系统性红斑狼疮继发性肾病综合征患者68例,为LNS组。同期选取本院健康体检正常者64例,为正常对照NC组。全自动生化分析仪检测各组血清Lp(a)和血脂等指标;酶联免疫吸附(Elisa)法测定血清PAI-1水平。结果:1与NC组比较,血清Lp(a)和PAI-1水平在PNS和LNS两组中均显著升高(P0.05),PNS组比LNS组升高更为明显,差异有统计学意义(P0.05);2LP(a)与PAI-1秩相关系数(rs)分析,在PNS组中r_s=0.328,P=0.006,LNS组中r_s=0.439,P=0.006;3二元logistic回归分析表明,LP(a)和PAI-1均是PNS和LNS的危险因素;4ROC曲线分析表明,血清Lp(a)、PAI-1对PNS和LNS诊断的ROC曲线下面积(AUC~(ROC))分别为0.895、0.874和0.848、0.813,两者联合检测对PNS和LNS诊断的AUC~(ROC)分别为0.947和0.919。结论:血清Lp(a)与PAI-1水平在PNS和LNS患者体内均明显升高,PNS患者升高更为显著;Lp(a)与PAI-1水平在PNS和LNS患者中均显著正相关;LP(a)和PAI-1均是PNS和LNS的危险因素,两者水平的变化与PNS和LNS的发生相关。联合检测Lp(a)与PAI-1水平对PNS和LNS的诊治具有一定的临床应用价值。     

Status of fibrinolysis in systemic lupus erythematosus

In patients with systemic lupus erythematosus (SLE) both a haemorrhagic diathesis and a tendency to thrombosis of the venous and arterial vessels can be observed. In the course of the disease, thrombosis of the leg or pelvic veins developed in 20 per cent of 188 patients. The levels of alpha 2-plasmin inhibitor, plasminogen, fibronectin and of factor VIII complex were increased in patients with SLE compared with a control group. Fifty per cent of the patients showed no increase in fibrinolytic activity after venous occlusion measured with the fibrin plate method. This suggests a reduced fibrinolytic capacity in SLE probably caused by alteration of the endothelial cells through immune complex vasculitis. In addition, the lupus anticoagulant and an acquired antithrombin III deficiency in nephrotic syndrome in SLE are to be considered thrombophilic mechanisms. In the individual case there is an overlapping of hyper- and hypocoagulability.     

Changes in serum level and affinity for concanavalin A of human alpha 1-proteinase inhibitor in severe burn patients: relationship to natural killer cell activity

In serum from five patients with severe burns, alpha 1-proteinase inhibitor (alpha 1-PI) was analyzed and then isolated by immunosorption chromatography. By Con A-Sepharose chromatography alpha 1-PI was separated into two types of fractions: the first containing the Con A-non-reactive isoforms and the second containing the Con A-reactive isoforms. The increase of alpha 1-PI serum level in burn patients is associated on the fifth day after the burn with a significant shift toward species enriched in bi-antennary oligosaccharides (Con A-reactive isoforms). This latter change passed very quickly and ten days after the burn, whereas the alpha 1-PI serum level was still high, the difference in proportions of Con A-reactive and non-reactive isoforms was not statistically significant. With respect to the difference in oligosaccharide structure, it appeared that the glycan moiety was involved in the inhibitory effect on natural killer cell activity. At the same concentration, purified alpha 1-PI and retained alpha 1-PI isoforms had an equal effect, whereas the non-retained alpha 1-PI isoforms were more efficient (P less than or equal to 0.01). Purified alpha 1-PI and its isoforms inhibited the natural killer cell activity in a dose-dependent manner.     

Changes in renal haemodynamics in the nephrotic syndrome

It has repeatedly been found that haemodynamic changes during hypoproteinaemia in the chronic phase of the nephrotic syndrome are different from those during hypoproteinaemia in the acute phase. In our series of patients, a decrease in the filtration fraction and relative hyperperfusion of the kidneys were associated with the presence of the nephrotic syndrome. No significant changes in renal haemodynamics were observed in patients with chronic glomerulonephritis without the nephrotic syndrome or in a group of healthy volunteers. The question of whether relative hyperperfusion of the kidneys in a repeatedly relapsing nephrotic syndrome can lead to the development of focal segmental glomerulosclerosis needs to be elucidated.