Urinary Thrombin as a Marker of Glomerular Inflammation Associated with Renal Injury in Type 2 Diabetes.

Glomerular inflammation is a putative aggravation factor for type 2 diabetic nephropathy and urinary thrombin is a novel marker of glomerular inflammation. To clarify the relationship between glomerular inflammation and progression of the nephropathy, we measured urinary thrombin in 118 patients with type 2 diabetic nephropathy at different stages. To investigate the implications of urinary thrombin in the nephropathy, we compared urinary thrombin with expression of tissue factor, the trigger of blood coagulation activation, in glomeruli and with markers of renal injury (estimated glomerular filtration rate (eGFR) and proteinuria). Urinary thrombin was found in 4.9% (3/61), 0.0% (0/12), 29.6% (8/27) and 50.0% (9/18) of patient groups at stages 1, 2, 3 and 4, respectively. Thus, urinary thrombin was negligible in the patients at early stages (stages 1 and 2), but was present predominantly in the patients at advanced stages (stages 3 and 4). Tissue factor was expressed in accumulated macrophages in glomeruli, which indicates that thrombin may be generated in inflamed glomeruli presumably via inflammation-induced activation of the exudated coagulation factors into glomerular tissues and then be excreted in urine. Urinary thrombin was significantly associated with both decreased eGFR and increased proteinuria in type 2 diabetic nephropathy. Therefore, increased urinary thrombin in patients with advanced stages of type 2 diabetic nephropathy suggests that glomerular inflammation may injure the tissues, thereby impairing renal function. Monitoring an effect of anti-diabetic treatments on glomerular inflammation in the patients with type 2 diabetic nephropathy may be a possible application of urinary thrombin.

Hypoelectrolytic isoosmotic solution for infusion prevents saline-induced ultrastuructural artifacts of renal biopsy specimens.

Artifacts in the process of specimen preparation are frequent in ultrastructural evaluation of renal biopsy. We hypothesized that the common practice of wrapping kidney biopsy specimens in saline-soaked gauze to prevent the drying of the specimens could be the major factor of artifacts. In this study, whole kidneys from two male Sprague-Dawley rats were used. Before fixation, fresh small cubes of kidney tissue were macerated in saline (Saline group) or hypoelectrolytic isoosmotic solution for infusion (HISI group) (Sorita T3 or SOLDEM 3A) for 10 or 30 min. Then, the specimens were processed by 1% OsO(4) in 0.1 M phosphate buffer (pH 7.4) and embedded by EPON 812 for ultramicroscopic analysis. In the Saline group, ultrastructural examination revealed swollen podocyte, swollen capillary protuberance of the mesangium into the glomerular capillary loop, tubular cells with swollen mitochondria and microvilli, and the smooth muscle cells in the arteriolar wall with marked vacuolar degeneration were detected after 10 min maceration in saline and these findings become more pronounced after 30 min maceration. However, in the HISI group, these artifacts were not identified or limited within 30 min. It is postulated that HISI solution could prevent the artifacts, and be used for soaking and wrapping instead of physiologic saline solution.

Urinary thrombin: a novel marker of glomerular inflammation for the diagnosis of crescentic glomerulonephritis (prospective observational study).

BACKGROUND: Crescentic glomerulonephritis (CresGN), an uncommon rapidly progressive disease, is characterized by severe glomerular inflammation with fibrin deposition. The lack of specific CresGN biomarkers delays diagnosis and threatens life. Because fibrin deposits in CresGN glomeruli indicate thrombin generation, we hypothesized that thrombin is excreted in urine and is a specific CresGN biomarker. METHODS: We measured urinary thrombin activity in 200 untreated patients (17 with CresGN, 183 with primary glomerulonephritis) and controls (8 patients with healed CresGN, 11 with nephrosclerosis, and 10 with tubulointerstitial nephritis, and 66 healthy volunteers). CresGN types included 15 pauci-immune and 2 immune complex. We assessed the diagnostic accuracy of thrombinuria in 169 patients with hematuria and proteinuria. Renal biopsy tissues were immunostained for tissue factor and fibrin. We analyzed the relationship of thrombinuria to plasma thrombin-antithrombin complex, hematuria, proteinuria, glomerular filtration rate, glomerular fibrin deposition, antineutrophil cytoplasmic antibodies (ANCAs), and C-reactive protein (CRP). We studied changes in thrombin activities after glucocorticoid treatment in 12 patients with thrombinuria. RESULTS: The highest thrombinuria occurrence was in CresGN (70.6%), followed by membranoproliferative glomerulonephritis (41.7%), IgA nephropathy (9.2%), and acute glomerulonephritis (0%). More than 75% of patients with nonproliferative glomerulonephritis manifested no thrombinuria. No controls had thrombinuria. Thrombinuria showed high CresGN specificity (90.1%) and moderate sensitivity (70.6%) and was detected in 4 of 7 patients with ANCA-negative CresGN. In CresGN, thrombinuria was associated with fibrin deposition in glomerular extracapillary tissue, where monocytes/macrophages expressed tissue factor. Thrombinuria in CresGN was unrelated to plasma thrombin-antithrombin complex, hematuria, proteinuria, glomerular filtration rate, and CRP. After glucocorticoid treatment, thrombinuria in patients with CresGN rapidly disappeared but proteinuria and hematuria persisted. CONCLUSIONS: Thrombinuria was specific for glomerular inflammation, was unaffected by systemic inflammation or coagulation, and demonstrated good diagnostic accuracy for CresGN including ANCA-negative cases. Thrombinuria measurement may provide risk-free diagnosis and screening for CresGN.

[Soluble fibrin is not excreted in urine and its plasma level is elevated in nephrotic syndrome].

Soluble fibrin (SF) is produced by activated blood coagulation reaction and is useful to diagnose thrombotic diseases. We measured plasma and urine SF levels in nephritic patients to assess the hypercoagulability state associated with the disease. Before they received anti-coagulation or anti-platelet therapies, 60 patients underwent measurement of plasma SF and D-dimer levels by Latex agglutination turbidimetric immnoassay (LA). Urinary SF levels were also measured by LA. Plasma and urinary thrombin antithrombin III complex (TAT) levels were measured by enzyme immunoassay (EIA). Plasma SF levels showed a good correlation with plasma TAT levels but only weak positive correlations were observed between plasma D-dimer and SF or TAT levels. Plasma SF and D-dimer levels were significantly higher in the Iatients with nephrotic-range hypoalbuminemia (< or =3 g/dL) than those without it. Contrarily there was no significant difference in plasma TAT levels between these two groups of patients. In almost all patients, urinary SF levels were under the detection limit. However, TAT was excreted into urine more frequently in patients showing the nephrotic range of hypoalbuminemia at 38.2% than in non-nephrotic patients at 8.0%. Thus, plasma SF levels more precisely indicate activated blood coagulation reaction than plasma TAT levels in nephrotic patients, probably because the plasma SF is not excreted into urine, while plasma TAT is.

Enteropeptidase, a type II transmembrane serine protease.

Enteropeptidase, a type II transmembrane serine protease, is localized to the brush border of the duodenal and jejunal mucosa. It is synthesized as a zymogen (proenteropeptidase) that requires activation by another protease, either trypsin or possibly duodenase. Active enteropeptidase then converts the pancreatic precursor, trypsinogen, to trypsin by cleavage of the specific trypsinogen activation peptide, Asp-Asp-Asp-Asp-Lys- Ile that is highly conserved in vertebrates. Trypsin, in turn, activates other digestive zymogens such as chymotrypsinogen, proelastase, procarboxypeptidase and prolipase in the lumen of the gut. The important biological function of enteropeptidase is highlighted by the manifestation of severe diarrhea, failure to thrive, hypoproteinemia and edema as a result of congenital deficiency of enteropeptidase activity in the gut. Conversely, duodenopancreatic reflux of proteolytically active enteropeptidase may cause acute and chronic pancreatitis.

Overexpression of TNNI3K, a cardiac-specific MAP kinase, promotes P19CL6-derived cardiac myogenesis and prevents myocardial infarction-induced injury.

TNNI3K is a new cardiac-specific MAP kinase whose gene is localized to 1p31.1 and that belongs to a tyrosine kinase-like branch in the kinase tree of the human genome. In the present study we investigated the role of TNNI3K in the cardiac myogenesis process and in the repair of ischemic injury. Pluripotent P19CL6 cells with or without transfection by pcDNA6-TNNI3K plasmid were used to induce differentiation into beating cardiomyocytes. TNNI3K promoted the differentiation process, judging from the increasing beating mass and increased number of alpha-actinin-positive cells. TNNI3K improved cardiac function by enhancing beating frequency and increasing the contractile force and epinephrine response of spontaneous action potentials without an increase of the single-cell size. TNNI3K suppressed phosphorylation of cardiac troponin I, annexin-V(+) cells, Bax protein, and p38/JNK-mediated apoptosis. Intramyocardial administration of TNNI3K-overexpressing P19CL6 cells in mice with myocardial infarction improved cardiac performance and attenuated ventricular remodeling compared with injection of wild-type P19CL6 cells. In conclusion, our study clearly indicates that TNNI3K promotes cardiomyogenesis, enhances cardiac performance, and protects the myocardium from ischemic injury by suppressing p38/JNK-mediated apoptosis. Therefore, modulation of TNNI3K activity would be a useful therapeutic approach for ischemic cardiac disease.

Urokinase-immobilization suppresses inflammatory responses to polyurethane tubes implanted in rabbit muscles.

Urokinase and plasmin appear to have antiinflammatory activity in some injury models, and urokinase immobilization has been clinically used to prevent thrombus formation in various implants, including intravenous indwelling catheters and subcutaneous drainage tubes. In the present study, polyurethane tubes were embedded in rabbit muscle for 3 months and the effect of urokinase immobilization on inflammatory responses to the implanted tubes was studied at 1 week, 1 month, and 3 months. Mononuclear leukocyte accumulation occurred around implanted polyurethane tubes and peaked after 1 month, but was reduced significantly by urokinase immobilization. The treatment also lessened as well as delayed eosinophil accumulation, but did not affect fibrosis caused by implanted tubes. These results indicate suppressive effects of urokinase immobilization on polyurethane-elicited inflammatory responses and suggest that an approach to develop persistently active urokinase immobilization is rational for successful long-term device implantation.

Protective effect of vascular endothelial growth factor/vascular permeability factor 165 and 121 on glomerular endothelial cell injury in the rat.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) promotes the repair of injured vessels by stimulating angiogenesis. VEGF/VPF reportedly has cytoprotective activity but no study has shown the protective effect of VEGF/VPF on glomerular endothelial cells. We examined whether recombinant VEGF/VPF121 and VEGF/VPF165 isoforms could prevent injury of glomerular endothelial cells. Mild glomerular injury was induced in rats by an intravenous-injection of a limited dose of anti-Thy-1.1 antibody to obtain lesions similar to those found in the human disease. Recombinant VEGF/VPF165, VEGF/VPF121 or BSA was administered 4 h before the injection of the antibody, and once daily for 3 days. In the BSA-injected rats, mesangial cell lysis and endothelial cell injury in dilated capillary tufts were evident without endothelial cell apoptosis on days 1-4. Thereafter, cell proliferation and repair began and remodeling of the glomeruli was completed by day 28. Macrophages but not polymorphonuclear leukocytes accumulated significantly in the glomeruli on days 1-4. Treatment with VEGF/VPF isoform protected endothelial cells but not mesangial cells from destruction on day 1, and accelerated the repair of both types of cells, which was completed by day 18, 10 days earlier than that of the control animals. The results indicate that VEGF/VPF121 or VEGF/VPF165 can protect glomerular endothelial cells against injury, independent of apoptosis-inhibition activity, thereby promoting reconstruction of glomeruli. The protective effect of VEGF/VPF on endothelial cells suggests that it could provide therapeutic benefit for certain kidney diseases.

[Evaluation of thrombin in urine as a real-time indicator of clotting activation in glomerulonephritis].

When tissues are injured and bleeding occurs, blood clotting is immediately activated and fibrin clots are formed by thrombin. Afterwards, antithrombin III promptly inactivates thrombin, which restricts the clotting to the bleeding site. In inflamed sites, tissue factor is expressed on cells in the lesion by stimulation from cytokines, and produces thrombin. In this case, thrombin may survive longer because of inefficient inactivation by antithrombin III due to dilution and less perturbation in the interstitial fluid, and therefore, has a greater chance to activate thrombin receptors (protease-activated receptors: PARs) on the cells, which induces various cellular events including proliferation, migration, and shape change. Recent studies have suggested a pathophysiological association of the PAR pathway with crescentic glomerulonephritis. However, the role of thrombin in human diseases has not been fully studied, probably because of a lack of simple and reliable methods for measuring thrombin in clinical samples. To solve this problem, we developed an ELISA system for human alpha-thrombin and applied it to the measurement of thrombin in the urine of patients with glomerulonephritis. Thrombin in urine was detected in glomerulonephritic patients but not in healthy volunteers or disseminated intravascular coagulation patients, which suggests that thrombin in urine may reflect thrombin generation by clotting activation in the glomerular lesion.

Assessment of thrombin in the urine of glomerulonephritic patients by enzyme-linked immunosorbent assay.

BACKGROUND: Accumulating evidence suggests that blood clotting occurs in inflamed glomeruli, although its role in the pathophysiology of glomerulonephritis remains to be elucidated. To address this issue, a simple and reliable method for evaluating clotting in glomeruli is necessary. Here, we developed an enzyme-linked immunosorbent assay (ELISA) for thrombin in urine to evaluate the degree of clotting activation in diseased glomeruli. METHODS: Monoclonal antibodies against human alpha-thrombin were raised and used for sandwich ELISA to measure thrombin. Thrombin was measured in urine samples from normal volunteers and from patients with glomerulonephritis or disseminated intravascular coagulation (DIC). RESULTS: Thrombin antigen was not detected in the urine of healthy volunteers or of patients with DIC, but was detected in the urine from two-thirds of glomerulonephritic patients. The average concentration in positive samples was 3.79 microg/L. Urinary thrombin concentrations measured by ELISA correlated well with thrombin activities measured by hydrolysis of a synthetic substrate. CONCLUSION: We suggest that thrombin antigen in urine measured by ELISA is not affected by systemic thrombin production in the vessels, and reflects blood clotting activation in glomerulonephritic lesions. A close relationship between urinary thrombin and glomerulonephritis indicates a possible involvement of clotting in disease development, and measurement of urinary thrombin may provide a real-time marker for monitoring renal diseases.

Expression of enteropeptidase in differentiated enterocytes, goblet cells, and the tumor cells in human duodenum.

Enteropeptidase (EP) is a serine proteinase and activates trypsinogen to trypsin, thus playing an important role in food digestion. Nevertheless, the localization of EP is still controversial, likely due to a lack of studies using specific antibodies against EP. The aim of this study was to define cellular localization of EP in human duodenum and expression in tumor cells at the duodenal region. Immunohistochemical staining for resected tissues was performed with two antibodies against recombinant EP light and heavy chains, respectively. In situ hybridization was done with two RNA probes that include either the light or the heavy chain sequences of proEP, respectively. The two antibodies reacted with enterocytes, accentuated on the brush border, and goblet cells, with increasing intensity from the bottom of crypts to the top of villi. Paneth cells, neuroendocrine cells, Brunner's glands, lymphocytes, smooth muscle, or connective tissue did not react with the antibodies. The two RNA probes detected EP mRNA expression only in enterocytes and goblet cells. EP is produced in enterocytes and goblet cells, and the localization on the brush border of the cells is reasonable for the physiological activation of digestive enzymes. Interestingly, the antibodies reacted with tumor cells in duodenal polyps and adenocarcinoma at the duodenum but not in Brunner's gland adenoma. EP seems to be a marker of differentiated enterocytes and goblet cells, which suggests the existence of a common progenitor of these cells. Furthermore, EP may be a useful marker of tumor cells originating from these cells.

Vascular permeability enhancement in solid tumor: various factors, mechanisms involved and its implications.

Most solid tumors are known to exhibit highly enhanced vascular permeability, similar to or more than the inflammatory tissues. Common denominators affecting both cancer and inflammatory lesions are now well known: bradykinin (BK), nitric oxide (NO), peroxynitrite (ONOO(-)), prostaglandins (PGs), collagenases or matrix metalloproteinases (MMPs) and others. Incidentally, enzymes involved in these mediator syntheses are upregulated or activated. Initially described vascular permeability factor (VPF) (proteinaceous) was later identified to be the same as vascular endothelial growth factor (VEGF), which promotes angiogenesis of cancer tissues as well. These mediators cross-talk or co-upregulate each other, such as BK-NO-PGs system. Therefore, vascular permeability observed in solid tumor may reflect the other side of the coin (angiogenesis). The vascular permeability and accumulation of plasma components in the interstitium described here is applicable for predominantly macromolecules (molecular weight, Mw>45 kDa), but not for low molecular compounds as most anticancer agents are. Macromolecular compounds (e.g., albumin, transferrin) or many biocompatible water-soluble polymers show this effect. Furthermore, they are not cleared rapidly from the sites of lesion (cancer/inflammatory tissue), thus, remain for prolonged time, usually for more than a few days. This phenomenon of "enhanced permeability and retention effect" observed in cancer tissue for macromolecules and lipids is coined "EPR effect", which is now widely accepted as a gold standard for anticancer drug designing to seek more cancer-selective targeting using macromolecular drugs. Consequently, drastic reduction of the systemic side effect is observed, while the macromolecular drugs will continuously exert antitumor activity. Other advantages of macromolecular drugs are also discussed.