Procoagulant activity in kidneys of normal and bacterial lipopolysaccharide-treated rabbits

Fibrin formation in the kidney is frequently associated with clinically-significant renal dysfunction. We therefore measured and characterized the procoagulant activity (PCA) which is present in normal kidneys and in kidneys of rabbits with the Shwartzman phenomenon induced by two injections of bacterial lipopolysaccharide (LPS; E. coli LPS 055:B5,25 micrograms/kg and 50 micrograms/kg administered 24 hrs apart with rabbits sacrificed 12 hrs after the second injection). PCA was measured in sonicated tissue by one-stage coagulation assay. In normal kidneys the amounts of PCA in the inner medulla, outer medulla and inner cortex were 18.2 +/- 3.2, 44.1 +/- 3.8 and 78.5 +/- 5.7 percent, respectively, of that in the outer cortex (N = 31). Glomeruli (purified by the iron oxide magnetic method to greater than 95 percent homogeneity) contained 21.6 +/- 8.8 arbitrary units/micrograms protein compared with tubular fragments which contained 13.9 +/- 2.6 U/micrograms protein (N = 9). In LPS-treated rabbits PCA (in units/micrograms) increased in outer cortex from 33.7 +/- 3.9 (control) to 73.4 +/- 10.4 (LPS, P less than 0.01), in inner cortex from 26.7 +/- 2.9 (control) to 83.3 +/- 17 (LPS, P less than 0.02), in outer medulla from 12.9 +/- 2.4 (control) to 54.5 +/- 16.5 (LPS, P less than 0.05), and in inner medulla from 12.2 +/- 2.4 (control) to 32.1 +/- 4.9 (LPS, P less than 0.01). Glomerular PCA increased from 21.6 +/- 8.8 (control) to 88.8 +/- 20.7 (LPS) units/micrograms (P = 0.01), while tubular fragment preparation PCA increased from 13.9 +/- 2.6 (control) to 44.6 +/- 12.7 (LPS) U/micrograms (P = 0.02) (N = 9 per group).(ABSTRACT TRUNCATED AT 250 WORDS)     

Procoagulant activity in renal transplant recipients

Procoagulant activity (PCA) of leukocytes of renal transplant recipients was studied. This material, which activates coagulation, has previously been shown to be released from macrophages after they interact with mitogen-stimulated or antigen-stimulated T lymphocytes. Under endotoxin-free conditions, PCA of peripheral blood leukocytes, incubated for 90 min in tissue culture, was elevated in postoperative transplant recipients and in many transplant patients tested around the time of a rejection episode. The response to lipopolysaccharide added during culture was also increased in these populations. The PCA response was factor-VII-dependent when tested with washed peripheral blood mononuclear cells (PBMC), but was factor-VII-independent when tested with unwashed PBMC in their original culture medium. The results indicate a possible link between immunologic events and coagulation in transplant recipients.     

Procoagulant activity in hemostasis and thrombosis: Virchow's triad revisited

Virchow's triad is traditionally invoked to explain pathophysiologic mechanisms leading to thrombosis, alleging concerted roles for abnormalities in blood composition, vessel wall components, and blood flow in the development of arterial and venous thrombosis. Given the tissue-specific bleeding observed in hemophilia patients, it may be instructive to consider the principles of Virchow's triad when investigating mechanisms operant in hemostatic disorders as well. Blood composition (the function of circulating blood cells and plasma proteins) is the most well studied component of the triad. For example, increased levels of plasma procoagulant proteins such as prothrombin and fibrinogen are established risk factors for thrombosis, whereas deficiencies in plasma factors VIII and IX result in bleeding (hemophilia A and B, respectively). Vessel wall (cellular) components contribute adhesion molecules that recruit circulating leukocytes and platelets to sites of vascular damage, tissue factor, which provides a procoagulant signal of vascular breach, and a surface upon which coagulation complexes are assembled. Blood flow is often characterized by 2 key variables: shear rate and shear stress. Shear rate affects several aspects of coagulation, including transport rates of platelets and plasma proteins to and from the injury site, platelet activation, and the kinetics of fibrin monomer formation and polymerization. Shear stress modulates adhesion rates of platelets and expression of adhesion molecules and procoagulant activity on endothelial cells lining the blood vessels. That no one abnormality in any component of Virchow's triad fully predicts coagulopathy a priori suggests coagulopathies are complex, multifactorial, and interactive. In this review, we focus on contributions of blood composition, vascular cells, and blood flow to hemostasis and thrombosis, and suggest that cross-talk among the 3 components of Virchow's triad is necessary for hemostasis and determines propensity for thrombosis or bleeding. Investigative models that permit interplay among these components are necessary to understand the operant pathophysiology, and effectively treat and prevent thrombotic and bleeding disorders.     

The influence of pH and urine composition on urease enzymatic activity in human urine

Summary It is reasonable to assume that the rate of pH increase in urine induced by urease-producing microorganisms is one of the factors which determine whether crystallisation with subsequent stone formation will occur or not. To evaluate how the time needed to increase urine pH varies between different urine samples and how it depends on urine composition, a standardised amount of urease was added to different human urine samples. The incubations were performed in a pH-stat. This allowed simultaneous study of how urease enzymatic activity depends on urine pH and how it varies between different urines. The enzymatic activity was found to be negatively correlated to urine pH and to vary between different urines. The rate of the pH increase varied markedly between different urines. Small pH increases depended on the native urine pH and urease enzymatic activity. Higher pH increases up to the levels of phosphate crystallisation depended more on urine phosphate, the major urine buffer. The results presented show that urine composition influences the urease-induced pH increase. This might have clinical implications.     

Neutral metalloproteinases in human urine from normal patients and renal disease patients

Summary: We examined the activity and the characteristics of gelatinolytic enzymes in the urine from normal subjects and the patients with glomerulonephritis (GN). Gelatinolytic activity was assayed by using [³H]gelatin (heat-denatured type I collagen) as a substrate. the activity found to be 2463 ± 204 U/day (mean ± SEM, n = 17) in the urine from the healthy individuals, but was negligible in plasma samples. In GN patients (n=24) urinary gelatinolytic activity (724± 149 U/day, P < 0.001) was significantly lower compared with the healthy individuals. Partial purification (anion exchange column chromatography followed by gel filtration) revealed that two active gelatinolytic enzymes (molecular weight [Mr]92 kDa; high molecular weight [HMW] gelatinase, Mr 40 kDa; low molecular weight [LMW] gelatinase) were present in the urine obtained from the healthy individuals. the activity of these enzymes was inhibited by EDTA, 1,10-phenanthroline and α₂-macroglobulin, but not by inhibitors for serine, cystein and aspartic proteinase. As the optimum pH was in the neutral range (pH 6–9), these gelatinases were considered to be neutral endometalloproteinases. These enzymes could degrade acid soluble type IV collagen and native rat glomerular basement membrane (GBM) in addition to gelatin, but not type I collagen. Immunoblotting with antibodies against human metalloproteinases (MP) identified HMW gelatinase with matrix metalloproteinase (MMP)-9 (gelatinase B) and LMW gelatinase with MMP-2 (gelatinase A), respectively. As these enzymes are metalloproteinase (MP) capable of degrading GBM and its component, urinary MP may be a useful subject as a tool searching the metabolic alteration of glomerular extracellular matrix in renal diseases.     

Association of urinary macromolecules with calcium oxalate crystals induced in vitro in normal human and rat urine

This study was undertaken to identify proteins which are found associated with calcium oxalate crystals induced in vitro in normal human and rat urine. Crystallization was initiated by adding sodium oxalate individually to each urine sample without centrifugation and filtration. Crystals were collected and analyzed by scanning electron microscopy and X-ray diffraction. Crystal matrix proteins (CMPs) were obtained by demineralization of the crystals with ethylenediaminetetra-acetic acid (EDTA) and analyzed by western blotting technique for immunological identification. Crystals produced in human urine were found to be a mixture of calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) while those produced in rat urine were exclusively COD. CMPs extracted from crystals in human urine comprised, in addition to prothrombin-related proteins, osteopontin and albumin. However, CMPs extracted from crystals in rat urine contained only osteopontin and albumin. Prothrombin-related proteins were found only in trace amounts. In a separate experiment, rat urine samples were supplemented with COM before inducing crystallization. Similar results were observed showing that CMP contained osteopontin, albumin and trace amounts of prothrombin-related proteins. We conclude that several urinary macromolecules including not only prothrombin-related proteins, but also osteopontin and albumin, become associated with CaOx crystals. The incorporation of these proteins in growing stones is not only due to the presence of -carboxyglutamic acid as it was suggested for prothrombin-related proteins, but may be due to other factors such as urinary chemistry, presence of glutamic and aspartic acid residues, and calcium-binding sites.     

A urine finding associated with obesity.

Corn starch can be identified by its physico-chemical propertyof producing Maltese cross birefringence under polarized light.In     

Human melanoma associated with 24 nm particles

Electron microscopic examination of epidermal tissue adjacent to a skin melanoma in a White male showed the presence of 24 nm particles within a cytoplasmic vacuole. No aetiological significance can be attributed to this single finding but it is possible that examination of tissues proximal to these neoplasms may be of value. As these tumors induce strong antibody responses in allogeneic and xenogeneic hosts it has often been suggested that they may be viral in origin.     

Biomarker discovery with SELDI-TOF MS in human urine associated with early renal injury: evaluation with computational analytical tools.

BACKGROUND: Urine proteomics is one of the key emerging technologies to discover new biomarkers for renal disease, which may be used in the early diagnosis, prognosis and treatment of patients. In the present study, we validated surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) for biomarker discovery in patients with mild ischaemic kidney injury. METHODS: We used first-morning mid-stream urine samples from healthy volunteers, and from intensive care unit patients we collected urine 12-24 h after coronary artery bypass graft (CABG) surgery. Samples of 50 volunteers were mixed to establish a reference sample (master pool). Urine samples were analysed with constant creatinine levels. RESULTS: The average intra- and interchip variation was found to be in the normal experimental range (CV of 10 to 30%). Computational analysis revealed (i) low intra-individual day-to-day variation in individual healthy volunteers; (ii) high concordance between the master pool sample and individual samples. Machine learning techniques for classification of CABG condition vs healthy patients showed that (iii) in the 3-20 kDa range, the joint activity of four protein peaks effectively discriminated the two classes, (iv) in the 20-70 kDa range, a single m/z marker was sufficient to achieve perfect separation. CONCLUSIONS: Our results substantiate the effectiveness of Seldi-TOF MS-based computational analysis as a tool for discovering potential biomarkers in urine samples associated with early renal injury.     

Variation of urate excretion with urine flow in normal man.

In normal persons, a decrease in urine flow following the injection of small amounts of vasopressin was accompanied by a significant decrease in the clearance and excretion of urate. When vasopressin and a small natriuretic dose of mannitol were administered together, urine flow and urate excretion again decreased. The recovery of urine flow to control values was accompanied by a similar recovery of urate excretion. Because a natriuretic dose of mannitol did not reverse the antiuricosuric effect of the flow decline, it is postulated that the flow effect probably reflects changes in a component of urate efflux distal to the loop of Henle.     

TLR4 (not TLR2) dominate cognate TLR activity associated with CoCrMo implant particles

Innate immune reactions to orthopedic implant debris are the primary cause of total joint replacement (TJR) failure over the long term (15–20 years). The role of pathogen associated pattern recognition receptors (i.e., TLRs) in regulating immune reactivity to metal implant particles remains controversial. Do different TLRs (i.e., TLR2 vs. TLR4) activated by their respective ligands in concert with metal implant debris elicit equivalent innate immune responses? In this investigation, our in vitro and in vivo data indicate that Gram‐negative PAMPs are more pro‐inflammatory than Gram‐positive PAMPs. In vitro results indicated TLR4 activation in concert with CoCrMo orthopedic implant debris (CoCrMo/LPS+) challenged primary macrophages resulted in significantly greater inflammatory responses than CoCrMo/PAM3CSK+ (TLR2). Similarly, in vivo results indicated CoCrMo/LPS+ TLR4 challenge induced a twofold increase in inflammation‐induced bone resorption (osteolysis) than CoCrMo/PAM3CSK+ (p < 0.01) or CoCrMo (p < 0.03) alone in an established murine calvaria model. This points to a more potent TLR4‐based effect of CoCrMo/LPS+ on innate immune responses, that is, IL‐1ß, TNF‐α, and resulting osteolysis. Differential CoCrMo/LPS+ induced osteolysis compared to CoCrMo/PAM3CSK+, reveals inherent differences in TLR4 versus TLR2 activation which are relevant to (i) how different types of implant debris elicit differential reactivity, (ii) how TLR2 Gram‐positive bacteria benefits from less immune activation possibly due to the down‐regulation of TLR2 surface expression, that subsequently impacts Gram‐positive infections in TJRs, and (iii) how using TLR4 LPS (a Gram‐negative agonist) may not accurately model Gram‐positive bacteria responses, alone and/or with specific types of implant particles, particularly CoCrMo alloy. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1007–1017, 2017.