Chitosan/copper nanocomposites: Correlation between electrical and antibacterial properties.

Correlation between electrical and antibacterial properties of chitosan/copper nanocomposites (CS/CuNPs) is investigated. We aim at achieving the minimum CuNPs concentration in a CS-matrix while keeping high antibacterial activity. UV-vis, TEM and XRD measurements confirms the formation of polygonal metallic CuNPs (ca. 30-50 nm). Interactions between NH2/OH groups of CS and CuNPs were determined by FTIR and XPS suggesting Cu chelation-induced mechanism during the CuNPs formation. DC electrical conductivity measurements reveals a percolation threshold at CuNPs volumetric concentration of ca. 0.143%. Antibacterial assays against Gram-positive bacteria and DC measurements helps correlate the antibacterial potency to the electron transfer between the negatively charged bacteria and CuNPs. Our study suggests that nanocomposite's maximum antibacterial activity is obtained below the electrical percolation threshold at extremely low CuNPs concentrations; this fact may prove useful in the design of nontoxic nanocomposites for biomedical applications.

Combined antibacterial/tissue regeneration response in thermal burns promoted by functional chitosan/silver nanocomposites.

We report the combined antibacterial/tissue regeneration responses to thermal burns promoted by functional chitosan/silver nanocomposites (CS/nAg) with ultralow silver content (0.018wt.%, 7-30nm). Our approach allows one to produce CS/nAg nanocomposites without silver nanoparticles (nAg) agglomeration, with bactericide potency higher than 1wt.% of nAg (ca. 10nm) content and, promoting the healing process in controlled thermal burns. CS/nAg films exhibit high antibacterial activity against S. aureus and P. aeruginosa after 1.5h of incubation, demonstrating the bacterial penetration into hydrated films and their interaction with nAg. Additionally, exceptional healing of induced thermal burns was obtained by increasing myofibroblasts, collagen remodeling, and blood vessel neoformation. These factors are associated with epiderma regeneration after 7days of treatment with no nAg release. Our results corroborate the controlled synthesis of nAg embedded in CS matrix with combined antibacterial/biocompatibility properties aiming to produce functional nanocomposites with potential use in wound dressing and health care applications.

Development of meniscus substitutes using a mixture of biocompatible polymers and extra cellular matrix components by electrospinning.

Despite the significant advances in the meniscus tissue engineering field, it is difficult to recreate the complex structure and organization of the collagenous matrix of the meniscus. In this work, we developed a meniscus prototype to be used as substitute or scaffold for the regeneration of the meniscal matrix, recreating the differential morphology of the meniscus by electrospinning. Synthetic biocompatible polymers were combined with the extracellular matrix component, collagen and used to replicate the meniscus. We studied the correlation between mechanical and structural properties of the polymer blend as a function of collagen concentration. Fibers were collected on a surface of a rapidly rotating precast mold, to accurately replicate each sectional morphology of the meniscus; different electro-tissues were produced. Detailed XRD analyses exhibited structural changes developed by electrospinning. We achieved to integrate all these electro-tissues to form a complete synthetic meniscus. Vascularization tests were performed to assess the potential use of our novel polymeric blend for promising meniscus regeneration.

Mechanisms for growth factor-induced pituitary tumor transforming gene-1 expression in pituitary folliculostellate TtT/GF cells.

PTTG1, a securin protein, also behaves as a transforming gene and is overexpressed in pituitary tumors. Because pituitary folliculostellate (FS) cells regulate pituitary tumor growth factors by paracrine mechanisms, epidermal growth factor (EGF) receptor (EGFR)-mediated PTTG1 expression and cell proliferation was tested in pituitary FS TtT/GF cells. EGFR ligands caused up to 3-fold induction of Pttg1 mRNA expression, enhanced proliferating cell nuclear antigen, and increased entry of G0/1-arrested cells into S-phase. PTTG binding factor mRNA expression was not altered. EGF-induced Pttg1 expression and cell proliferation was abolished by preincubation of TtT/GF cells with EGFR inhibitors AG1478 and gefitinib. Phosphatidylinositol 3 kinase, protein kinase C, and MAPK, but not c-Jun N-terminal kinase and Janus activating kinase signaling regulated EGF-induced Pttg1, as well as proliferating cell nuclear antigen mRNA expression and entry into S-phase. EGF-induced EGFR and ERK1/2 phosphorylation was followed by rapid MAPK kinase/ERK kinase-dependent activation of Elk-1 and c-Fos. EGF-induced Pttg1 expression peaked at the S-G2 transition and declined thereafter. Pttg1 cell cycle dependency was confirmed by suppression of EGF-induced Pttg1 mRNA by blockade of cells in early S-phase. The results show that PTTG1 and its binding protein PTTG binding factor are expressed in pituitary FS TtT/GF cells. EGFR ligands induce PTTG1 and regulate S-phase, mediated by phosphatidylinositol 3 kinase, protein kinase C, and MAPK pathways. PTTG1 is therefore a target for EGFR-mediated paracrine regulation of pituitary cell growth.

Na+-K+-ATPase inhibitors and renin release: relationship to calcium.

The inhibition of renin secretion and the vasoconstrictive action of cardiac glycosides may be attributed to increases in cytosolic calcium as a result of inhibition of Na+-K+-ATPase. These studies examined in the dog in vivo the role of calcium on the renal actions of ouabain as assessed from the modifying effects of calcium channel blockers. Since vanadate, another Na+-K+-ATPase, inhibitor, enhances in vitro the binding of ouabain to Na+-K+-ATPase, we examined the capacity of vanadate to modify the renal effects of ouabain in vivo. Infusion of ouabain (1 microgram X kg-1 X min-1) into the renal artery decreased RBF, GFR, and renin secretion, and produced diuresis and natriuresis. When ouabain was infused in dogs receiving the calcium channel blocker verapamil (100 microgram/min), it failed to suppress renin secretion or cause renal vasoconstriction. In addition, verapamil produced diuresis and natriuresis, which were greatly enhanced by ouabain (e.g., verapamil FENa 12.0 +/- 1.1----34.2 +/- 5.1%). The data strongly suggest that calcium entry into cells is a major mediator of the renin inhibitory effect and of the renal vasoconstriction induced by cardiac glycosides. The natriuresis observed during the calcium channel blocker infusion suggests that this drug may have a direct tubular effect on sodium reabsorption. Superimposition of vanadate (0.5 mumol/min) on ouabain infusion led to massive natriuresis (FENa, 5 +/- 1----35 +/- 4%), renal vasodilation (RBF 90 +/- 12----170 +/- 15 ml/min), and an increase in renin secretion (delta, 100%).(ABSTRACT TRUNCATED AT 250 WORDS)

Critical role of extracellular calcium in vanadate-induced renal vasoconstriction.

Intra-arterial infusion of vanadate (VO4) in dogs produces a reduction in renal blood flow (RBF), glomerular filtration rate (GFR), urine flow (V), and the fractional excretion of sodium (FENa+). To evaluate the role of Ca2+ in these changes VO4 was infused into the renal artery in the presence of the calcium antagonists trifluoperazine (TFP), verapamil, or EGTA. TFP inhibited the effect of VO4 on RBF (TFP + VO4:64.1, VO4:38.5 ml/min; P less than 0.05), GFR (TFP + VO4:22.9, VO4:9.3, ml/min; P less than 0.05) and V (TFP + VO4: 0.80, VO4: 0.38 ml/min; P less than 0.05) without changing FENa+ (TFP + VO4: 3.8, VO4: 3.2%). Similar changes were obtained with verapamil as well as with EGTA. Furthermore thyroparathyroidectomy (TPTX) decreased serum calcium (control: 8.78, TPTX: 4.98 mg/100 ml; P less than 0.05) and blunted the effects of VO4 on renal hemodynamics. Reestablishing normal serum Ca2+ by an intra-arterial infusion of CaCl2 elicited the VO4 effects of vasoconstriction and decreased GFR; V was not affected and FENa+ rose. The data support the idea that influx of extracellular calcium into smooth muscle cells mediates the hemodynamic effects of VO4 in the dog.

Carbonic anhydrase independent bicarbonate reabsorption in rats with chronic papillary necrosis.

The present study was designed to indirectly localize the tubular sites of carbonic anhydrase independent bicarbonate reabsorption in the rat. Papillary necrosis was induced in rats by intravenous administration of bromoethyleneamine hydrobromide (BEA) 6 weeks prior to the study, in order to assess the role of deep nephrons in this process. Acetazolamide alone, acetazolamide plus amiloride, and acetazolamide, amiloride plus furosemide were infused into rats with intact papillae (groups I, III, V) and rats with BEA-induced papillary necrosis (groups II, IV, VI). Our results show that chronic papillary necrosis does not alter carbonic anhydrase independent bicarbonate reabsorption, since the fractional excretion of bicarbonate (FEHCO3) was not significantly higher when acetazolamide was infused into animals with BEA-induced papillary necrosis as compared to those rats with intact papillae (FEHCO3 group I vs. group II: NS). The addition of amiloride hydrochloride, a blocker of distal acidification at the administered doses, increased FEHCO3 significantly in both, animals with intact papillae and those with papillary necrosis, to a similar degree. The addition of furosemide to acetazolamide and amiloride further induced a significant increase in FEHCO3 only in the group of animals with papillary necrosis (FEHCO3 group V 43.0 +/- 2.9% vs. group VI 52.1 +/- 0.9%; p less than 0.05). It appears from our study that deeper nephrons and papillary structures are not indispensable for carbonic anhydrase independent bicarbonate reabsorption in the rat on a chronic basis. The cortical collecting duct appears to have a significant capacity to reabsorb bicarbonate independent of carbonic anhydrase which can be blocked by amiloride.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal Na+-K+-ATPase in renin release.

The effects of ouabain and furosemide on renin secretion, renal function, and renal Na+-K+-ATPase were investigated in anesthetized dogs. Furosemide (2 mg/kg) induced significant diuresis, natriuresis, an increase in renal blood flow (RBF), and a fivefold increase in renin secretory rate (RSR), but no changes in glomerular filtration rate (GFR). Infusion of ouabain (1 microgram . kg-1 . min-1) into one renal artery during furosemide diuresis increased fractional sodium excretion from 22 +/- 2 to 30 +/- 3% from the ipsilateral kidney but did not change urine flow, RBF, or GFR, whereas RSR fell to control values (698 +/- 203 to 137 +/- 43). When ouabain preceded furosemide, the rise in RBF and RSR induced by furosemide was abolished but sodium excretion increased. Ouabain infused in vivo inhibited Na+-K+-ATPase in microsomal fractions from cortex (34%) and medulla (27%) as compared with control. Neither saline nor furosemide exerted any effect on Na+-K+-ATPase. Moreover, the effect of ouabain alone on Na+-K+-ATPase was not different from that of ouabain plus furosemide. No changes in Mg2+-ATPase were detected in any of the experiments. These results indicate that inhibition of renal Na+-K+-ATPase abolishes furosemide-induced renin secretion despite potentiation of the natriuretic effect of the diuretic. It is apparent that the level of activity of Na+-K+-ATPase is of prime importance for renin secretion. In addition, ouabain may act directly on the juxtaglomerular cells to inhibit renin secretion.

Distal acidification in the rabbit: role of diet and blood pH.

A distal acidification defect is said to exist in rabbits because this species does not achieve a normal urine minus blood (U-B) PCO2 gradient in response to sodium bicarbonate infusion. This observation contrasts with data derived from studies in isolated rabbit cortical collecting tubules that have shown an acidifying capacity when the tubules were obtained from acidotic animals. The present study was designed to examine the role of diet and blood pH on distal acidification in the rabbit. Maximal alkalinization of the urine by acute sodium bicarbonate infusion was associated with a low U-B PCO2 gradient (0.7 +/- 2.1 mmHg). Rabbits made acidotic by ammonium chloride administration for 1 wk achieved a substantial U-B PCO2 gradient (29 +/- 5 mmHg) in response to neutral sodium phosphate infusion. To further evaluate the role of blood pH on the ability to raise U-B PCO2 gradient, rabbits and rats made acidotic by chronic ammonium chloride administration were studied. Neutral sodium phosphate was then infused to stimulate distal acidification. At comparable levels of urinary phosphate concentration and blood pH, acidotic rabbits were able to achieve a U-B PCO2 (50 +/- 7 mmHg) comparable with that of acidotic rats (48 +/- 8.3 mmHg). These data show that the failure of rabbits to raise U-B PCO2 gradient can be partially corrected by prior exposure to acid in the diet and further corrected by maintaining the blood pH within the acidotic range.

Effect of sodium orthovanadate on renal renin secretion in vivo.

The effect of vanadate (0.5 mumol/min) on renin secretory rate (RSR) of the kidney has been studied in nembutal-anesthetized, volume-expanded dogs. Intrarenal vanadate infusion caused a 69.3 +/- 8.8% decrease in RSR. This was accompanied by marked decreases in renal blood flow (RBF), glomerular filtration rate (GFR) and fractional excretion of sodium (FENa). Renal vascular resistance rose from 1.3 +/- 0.09 to 6.1 +/- 2.3 mm Hg/ml/min (P less than .0005). Papaverine infusion partially blunted the effect of vanadate on RSR (RSR only fell to 42. +/- 10% of basal values). The decreases in RBF and GFR were also less and FENa slightly higher than normal. Acetylcholine prevented the effects of vanadate more fully. There was no fall in RBF, GFR or FENa and it basically abolished the fall in RSR which fell only 19.4 +/- 25.3 of control (P = N.S.). Nifedipine (a slow Ca++ channels blocker) also prevented the fall in RBF, GFR and FENa induced by vanadate. RSR did not change significantly (7.8 +/- 10.9%). These results clearly demonstrate that vanadate is a potent inhibitor of renin secretion and suggest that inhibition of smooth muscle Na+, K+, adenasine triphosphatase and changes in the cystosolic concentration of Na and Ca are involved in its mechanism. Changes in perfusion pressure and sodium delivery to the macula densa appear to have little if any role in the inhibition.

Metabolic and hormonal factors influencing extrarenal buffering of an acute acid load.

This study evaluates metabolic and hormonal factors influencing extrarenal buffering of an acute acid load. Phosphate deprivation of 2 weeks duration was associated with enhanced extrarenal acid buffering. The enhanced extrarenal buffering capacity of phosphate deprivation was not dependent on the presence of parathyroid glands. Parathyroid hormone administration to phosphate-deprived rats promoted a further enhancement of the buffering capacity of an acid load. Blood pH and HCO3 during acid loading were not significantly different between control and diphosphonate-treated rats and between phosphate-deprived rats and phosphate-deprived rats treated with diphosphonate. The mortality rate, however, was significantly higher in diphosphonate-treated rats than in rats not receiving the drug suggesting that diphosphonate blunts the buffering of an acid load in both control and phosphate-deprived rats. Chronic vitamin D administration and acute administration of arginine vasopressin in pharmacologic doses were associated with significant enhancement of buffering capacity as compared to control rats. Thyrocalcitonin administration to intact but not thyroparathyroidectomized rats was associated with diminished capacity to buffer an acid load. These data demonstrate that the buffering of an acute acid load is influenced by a number of dietary and hormonal factors probably acting at the level of the bone.

The effects of chronic papillary necrosis on acid excretion.

Complete papillary necrosis in rats can be induced within 1 month following a single injection of 2-bromoethylamine hydrobromide (BEA) (50 mg, i.v.). Utilizing a combination of clearance and balance techniques the effects of complete absence of the papilla was examined as regards urinary acidification, whole kidney glomerular filtration rate (GFR), single nephron GFR, and morphology. Whole kidney GFR was not different from control, however, the percent filtering juxtamedullary nephrons was markedly diminished (87.2 +/- 2.1 vs. 31.5 +/- 3.6% filtering, control vs. BEA, respectively, P less than 0.001) and significantly reduced in the superficial nephrons (80.6 +/- 3.6 vs. 62.2 +/- 6.1% filtering, control vs. BEA, respectively, P less than 0.05). There was a significant decrease in juxtamedullary single nephron GFR and an increase in the superficial single nephron GFR as assessed by the quantitative Hanssen's technique in the animals with chronic papillary necrosis. Complete papillary necrosis was associated with normal arterial bicarbonate concentration, pH, and plasma electrolyte concentrations. At the same degree of acidemia (induced by NH4Cl administration) minimal urinary pH, ammonium excretion, and titratable acid excretion were not different than seen in age matched controls. The response to Na2SO4 infusion and phosphate infusion was the same in both groups of animals. The urine-blood (U-B)pCO2, and index of urinary acidification, was identical in BEA and control animals. Scanning electron microscopy showed scarring of the juxtamedullary glomeruli one month after BEA. The papilla was sloughed and lying free in the renal pelvis in every experimental animal. These data demonstrate that complete papillary necrosis is not associated with acidosis nor a defect in urinary acidification.

Parathyroid hormone and extrarenal acid buffering.

The role of parathyroid hormone (PTH) on the extrarenal buffering of an acid load was examined during HCl infusion (5 meq x kg-1 x h-1) to bilaterally nephrectomized rats. Thyroparathyroidectomized (TPTX) rats replaced with PTH had significantly higher blood pH and HCO3 values than TPTX rats not infused with PTH. Administration of EDTA, in a dose shown to release PTH, was associated with a significant increase in buffering capacity in intact but not in TPTX rats. Colchicine, given in a dose capable of stimulating PTH release, was also associated with enhanced buffering capacity in intact but not in TPTX rats. In TPTX rats infused with acetazolamide and PTH, the hormone failed to enhance extrarenal buffering of an acid load. Animals with chronic renal failure, induced by infarction of the kidney, also had an enhanced capacity to buffer an acid load. This enhanced buffering capacity in chronic renal failure was abolished by TPTX. Acute renal failure induced by bilateral ureteral ligation was also associated with increased buffering only in the presence of parathyroid glands. These data demonstrated that PTH, from either an exogenous or endogenous source, enhances extrarenal buffering capacity of an acid load. Chronic and acute renal failure are associated with increased buffering capacity, which is dependent on the presence of parathyroid glands. The data suggest that this effect is mediated through carbonic anhydrase.