Electron-microscopic and immunocytochemical analyses of Weibel-Palade bodies in the human umbilical vein during pregnancy

Summary The present study was done to elucidate the biological significance of the Weibel-Palade body of human umbilical vein endothelial cells. Quantitative determinations of these endothelial-specific granules throughout pregnancy revealed that their numbers and size per cell profile were maintained at low levels from 12 to 19 weeks of gestation; then both rapidly increased from 33 weeks to full term. This increase coincided with the development of the rough endoplasmic reticulum and an increase in the number of endothelial cell pinocytotic vesicles. Light-microscopic peroxidase anti-peroxidase and electron-microscopic protein A-gold techniques provided evidence that factor VIII-related antigen was localized in the Weibel-Palade bodies. Furthermore, in vitro treatment of incubated umbilical vein tissue with compound 48/80, a histamine releaser, induced degranulation of Weibel-Palade bodies from the endothelium. The present study indicates that Weibel-Palade bodies are storage sites of both histamine and factor VIII-related antigen and have an important role in the obliteration of this vessel.     

A novel array-based assay of in situ tissue transglutaminase activity in human umbilical vein endothelial cells

Transglutaminases (TGs), a family of calcium-dependent transamidating enzymes, are involved in functions such as apoptosis and inflammation and play a role in autoimmune diseases and neurodegenerative disorders. In this study, we describe a novel array-based approach to rapidly determine in situ TG activity in human umbilical vein endothelial cells and J82 human bladder carcinoma cells. Amine arrays were fabricated by immobilizing 3-aminopropyltrimethoxysilane on glass slides. The assay was specific and highly reproducible. The average coefficient of variation betweens spots was 2.6% (n = 3 arrays), and the average correlation coefficients between arrays and between arrays/reactions were 0.998 and 0.976, respectively (n = 3 arrays). The assay was successfully applied to detect changes in TG activity induced by maitotoxin and to analyze inhibition of the TG activation with cystamine and monodansyl cadaverine. In addition, the assay demonstrated that intracellular reactive oxygen species regulate the maitotoxin-induced activation of TG. Thus, the array-based in situ TG activity assay constitutes a rapid and high-throughput approach to investigating the roles of TGs in cell signaling.     

VEGF-C attenuates renal damage in salt-sensitive hypertension

Salt-sensitive hypertension is a major risk factor for renal impairment leading to chronic kidney disease. High-salt diet leads to hypertonic skin interstitial volume retention enhancing the activation of the tonicity-responsive enhancer-binding protein (TonEBP) within macrophages leading to vascular endothelial growth factor C (VEGF-C) secretion and NOS3 modulation. This promotes skin lymphangiogenesis and blood pressure regulation. Whether VEGF-C administration enhances renal and skin lymphangiogenesis and attenuates renal damage in salt-sensitive hypertension remains to be elucidated. Hypertension was induced in BALB/c mice by a high-salt diet. VEGF-C was administered subcutaneously to high-salt-treated mice as well as control animals. Analyses of kidney injury, inflammation, fibrosis, and biochemical markers were performed in vivo. VEGF-C reduced plasma inflammatory markers in salt-treated mice. In addition, VEGF-C exhibited a renal anti-inflammatory effect with the induction of macrophage M2 phenotype, followed by reductions in interstitial fibrosis. Antioxidant enzymes within the kidney as well as urinary RNA/DNA damage markers were all revelatory of abolished oxidative stress under VEGF-C. Furthermore, VEGF-C decreased the urinary albumin/creatinine ratio and blood pressure as well as glomerular and tubular damages. These improvements were associated with enhanced TonEBP, NOS3, and lymphangiogenesis within the kidney and skin. Our data show that VEGF-C administration plays a major role in preserving renal histology and reducing blood pressure. VEGF-C might constitute an interesting potential therapeutic target for improving renal remodeling in salt-sensitive hypertension.     

Application of Dolichos biflorus in immunoassay detection of kidney collecting duct biomarkers

Currently there are no biomarkers for detecting collecting duct damage in man. Antibodies to several collecting duct-specific antigens exist but sandwich assays have been difficult to establish due to the need for two different antibodies to the same protein. We hypothesized that a collecting duct-specific lectin could be used in combination with a collecting duct-specific antibody to negate the need for two different antibodies. The collecting duct specificity of selected antibodies (NiCa II 13C2, Pap XI 3C7, HuPaP VII 2B11 and aquaporin 2), was verified by immunohistochemistry. Aquaporin 2 and Pap XI 3C7 were used successfully in setting up assays with the lectin Dolichos biflorus, using the Meso Scale Discovery (MSD) platform. Antigen expression was highest in the papillae of rat and human kidney (corresponding to the greatest density of collecting ducts) and was also present in normal urine. We propose that further qualification and validation would lead to an assay for detecting collecting duct damage in man.     

Radiofrequency Catheter Ablation Of Atrioventricular Nodal Reentry Tachycardia In A Patient With Inferior Vena Cava Anomaly

Curative radiofrequency catheter modification of the slow pathway is the recommended therapy for patients suffering from recurrent symptomatic atrioventricular nodal reentry tachycardia. This is usually performed via femoral vein and the inferior vena cava (IVC). Presence of venous occlusion or complex venous anomaly involving the IVC may preclude this approach. Here, we report a case with a complex venous anomaly involving the inferior vena cava, who underwent electrophysiological study and successful radiofrequency ablation by an alternative approach.     

Influence of cadmium on the distribution of the essential trace elements zinc and copper in the liver and kidneys of rats

Cd induced changes of Zn and Cd distribution in the liver and kidneys were studied in relation to Cd metallothionein (MT) synthesis. Wistar male rats were given CdCl₂ by sc injection of .8, 1.5, and 3.0 mg Cd/kg three times a week for three weeks. Cd levels of liver and kidneys increased with the increment of Cd dosage and 80–90% of Cd was found in the cytosol. The MT fractions contained 80–89% cytosolic Cd in the liver and 55–75% Cd in the kidneys. Zn concentrations in the liver increased following Cd administration, But Zn in the kidneys showed only slight increase. There was a distinct decrease of Cu concentration in the liver of the 3.0 mg group. In contrast, Cu concentrations in the kidneys increased about three times in the .8 and 1.5 mg Cd groups, but Cu in the 3.0 mg group showed only 1.5 times increase. The changes of these metal concentrations were observed mainly in the cytosol. Non-MT-Cd in the kidneys was maximum in the 1.5 mg group, but the 3.0 mg group showed significant decrease. In parallel with this decrease of Cd, Cu and Zn in the kidneys showed similar decrease. When the kidneys are injured, Zn and Cu appear to leak from this organ.     

Ferric cacodylate efficiently stimulates growth of rat renal glomerular epithelial cells in vitro

Rat renal glomerular epithelial cells (SGE1 cell line) can be maintained and grown continuously in serum-free medium supplemented with insulin, iron-saturated transferrin (Tr), selenium, bovine serum albumin (BSA), linoleic acid, and epidermal growth factor (EGF). Of the growth supplements used, Tr is essential for proliferation of the cells. In the present study, we describe the use of a unique iron-chelate complex, ferric cacodylate (Fe-Cac), positively charged molecules in neutral buffer, that could almost replace Tr in serum-free culture. It even stimulated the growth of SGE1 cells more efficiently than ferric chloride (FeCl₃) and other iron-chelate complexes, such as ferric nitrilotriacetate (Fe-NTA) and ferric citrate (Fe-Cit). The growth-stimulatory activity of Fe-Cac was exerted at iron concentrations of more than 0.01 g/ml, whereas a 10-fold excess of iron concentration was required with FeCl₃, Fe-NTA and Fe-Cit. We observed that SGE1 cells grew until confluent, then formed hemicysts (domes) in serum-free medium containing Fe-Cac, suggesting that Fe-Cac did not merely permit cell growth but also supported polarization and organization of the cells into a functional epithelial architecture. Moreover, since the stimulatory activity of Fe-Cac was completely abolished by desferrioxamine, a strong iron chelator, it is suggested that iron is crucial for growth of SGE1 cells. When the cells were treated with suramin, an inhibitor of cellular pinocytosis and endocytosis of a large spectrum of ligands including receptor-bound growth factors, growth-stimulatory activity of Tr was inhibited, whereas the activity of Fe-Cac was not affected. These results, taken together, strongly suggest that the growth-stimulatory activity of Fe-Cac is associated with iron delivery into the cells through the cell membrane by diffusion, which is different from Tr receptor-mediated endocytosis. The use of Fe-Cac for investigating iron-regulated cell proliferation is suggested.     

Successful treatment for perinephric abscess with recombinant human granulocyte colony-stimulating factor following nephrectomy in a patient of myelodysplastic syndrome: A case report

The 35-year-old man with myelodysplastic syndrome (MDS) and granulocytopenia with dry cough and high fever was eventually found to have a left perinephric abscess ofStaphylococcus aureus. He underwent left nephrectomy and drainage of perinephric space in conjunction with appropriate antibiotics. However, because of persistent granulocytopenia,Staph. aureus never cleared up with formation of only poor granulation. Recombinant human granulocyte colony-stimulating factor (G-CSF) was added to the above treatment leading to prompt improvement in granulocytopenia and emergence of the good granulation tissue. G-CSF will probably become one of the important agents in treating MDS with granulocytopenia.     

Metabolic adaptation of the renal carbohydrate metabolism. I. Effects of starvation on the gluconeogenic and glycolytic fluxes in the proximal and distal renal tubules

Summary The influence of starvation on renal carbohydrate metabolism was studied in the proximal and distal fragments of the nephron. Starvation induced a double and opposite adaptation mechanism in both fractions of the renal tubule. In renal proximal tubules, the gluconeogenic flux was stimulated progressively during a period of 48 hours of starvation (2.15 fold), due, in part, to a significant increase in the fructose 1,6-bisphosphatase and phosphoenolpyruvate carboxykinase activities although with different characteristics. Fructose 1,6-bisphosphatase activity from this tubular fragment increased only at subsaturating subtrate concentration (68%) which involved a significant decrease in the Km (35%) for fructose 1,6-bisphosphate while there was no change in Vmax. This behaviour clearly indicates that it is related to modifications in the activity of the preexistent enzyme in the cell. Proximal phosphoenolpyruvate carboxykinase activity increased proportionally at both substrate concentrations (86 and 89% respectively) which brought about changes in Vmax without changes in Kin, all of which are in accordance with variations in the cellular levels of the enzyme. In the renal distal tubules, the glycolytic capacity drastically decreased throughout the starvation time. At 48 hours 65% of inhibition was shown. We have found a short term regulation of phosphofructokinase activity by starvation which involves an increase in Km (2.2 fold) without changes in Vmax, as a result of these kinetic changes, an inactivation of phosphofructokinase was detected at subsaturating concentration of fructose 6-phosphate. On the contrary, this nutritional state did not modify the kinetic behaviour of renal pyruvate kinase. Finally, neither proximal glycolytic nor distal gluconeogenic capacities and related enzymes activities were changed during starvation.