Quantitative assessment of urea generation and elimination in healthy dogs and in dogs with chronic kidney disease

BACKGROUND: Kinetic assessment of urea, the main end product of protein metabolism, could serve to assess protein catabolism in dogs with chronic kidney disease (CKD). Protein malnutrition and catabolism are poorly documented in CKD and they often are neglected clinically because of a lack of appropriate evaluation tools. HYPOTHESIS: Generation and excretion of urea are altered in dogs with CKD. ANIMALS: Nine dogs with spontaneous CKD (IRIS stages 2-4) and 5 healthy research dogs. METHODS: Endogenous renal clearance (Clrenal) of urea and creatinine was measured first. Exogenous plasma clearance (Clplasma, total body clearance) of the 2 markers then was determined by an IV infusion of urea (250-1,000 mg/kg over 20 minutes) and an IV bolus of creatinine (40 mg/kg). Extrarenal clearance (Clextra) was defined as the difference between Clplasma)and Clrenal. Endogenous urea generation was computed assuming steady-state conditions. RESULTS: Median Clrenal and Clextra of urea were 2.17 and 0.21 mL/min/kg in healthy dogs and 0.37 and 0.28 mL/min/kg in CKD dogs. The proportion of urea cleared by extrarenal route was markedly higher in dogs with glomerular filtration rate<1 mL/kg/min than in normal dogs, reaching up to 85% of the total clearance. A comparable pattern was observed for creatinine excretion, except in 1 dog, Clextra remained<20% of Clplasma. CONCLUSION: Extrarenal pathways of urea excretion are predominant in dogs with advanced CKD and justify exploring adjunctive therapies based on enteric nitrogen excretion in dogs. A trend toward increased urea generation may indicate increased catabolism in advanced CKD.

The effect of different levels of dietary crude protein on urea metabolism of rusa deer (Cervus timorensis)

Two experiments were conducted to measure urea recycling and rumen flow dynamics in young rusa deer fed low (LP) or high (HP) protein diets. Pool size and flux rate of labelled urea. into and out of the blood pool were measured using single intravenous (i.v.) injection solutions containing [C-14] - and [N-15]-urea. A curve peeling technique was used to fit the enrichment of N-15 or specific radioactivity (SRA) of C-14 to exponential equations. Body urea-N pool size was significantly greater (P < 0.05) when a HP, compared to a LP diet, was fed. Urea space, expressed as a percent of live weight, total flux rate of urea through the blood pool and the irreversible loss of urea was similar for both diets. The mean (+/- S.E.M.) concentration of plasma urea-N was greater when animals were fed the HP diet compared to the LP diet (2 1.1 +/- 0.3 versus 14.4 +/- 1.4 mg/100 ml, respectively). Voluntary feed intake and digestibility of dietary components were also measured. Daily dry matter intakes were not affected by the crude protein (CP) content of the diet, although apparent DM digestibility was significantly greater for HP diet fed in both experiments. An intraruminal infusion of CrEDTA was used to determine rumen flow dynamics. Ruminal mean retention time, relative net outflow rate of water and passage rate constant (k(w)) were significantly greater (P < 0.05) when the HP diet was fed compared to the LP diet. The extent of urea metabolism and flux rates of urea between the blood and secondary pools appear similar to those previously reported for other ruminants fed diets contrasting in CP content. (c) 2005 Elsevier B.V. All rights reserved.

Effect of endogenous hydrogen sulfide inhibition on structural and functional renal disturbances induced by gentamicin

Animal models of gentamicin nephrotoxicity present acute tubular necrosis associated with inflammation, which can contribute to intensify the renal damage. Hydrogen sulfide (H2S) is a signaling molecule involved in inflammation. We evaluated the effect of DL-propargylglycine (PAG), an inhibitor of endogenous H2S formation, on the renal damage induced by gentamicin. Male Wistar rats (N = 8) were injected with 40 mg/kg gentamicin (im) twice a day for 9 days, some of them also received PAG (N = 8, 10 mg·kg-1·day-1, ip). Control rats (N = 6) were treated with saline or PAG only (N = 4). Twenty-four-hour urine samples were collected one day after the end of these treatments, blood samples were collected, the animals were sacrificed, and the kidneys were removed for quantification of H2S formation and histological and immunohistochemical studies. Gentamicin-treated rats presented higher sodium and potassium fractional excretion, increased plasma creatinine [4.06 (3.00; 5.87) mg%] and urea levels, a greater number of macrophages/monocytes, and a higher score for tubular interstitial lesions [3.50 (3.00; 4.00)] in the renal cortex. These changes were associated with increased H2S formation in the kidneys from gentamicin-treated rats (230.60 ± 38.62 µg·mg protein-1·h-1) compared to control (21.12 ± 1.63) and PAG (11.44 ± 3.08). Treatment with PAG reduced this increase (171.60 ± 18.34), the disturbances in plasma creatinine levels [2.20 (1.92; 4.60) mg%], macrophage infiltration, and score for tubular interstitial lesions [2.00 (2.00; 3.00)]. However, PAG did not interfere with the increase in fractional sodium excretion provoked by gentamicin. The protective effect of PAG on gentamicin nephrotoxicity was related, at least in part, to decreased H2S formation.

Effect of endogenous hydrogen sulfide inhibition on structural and functional renal disturbances induced by gentamicin

Animal models of gentamicin nephrotoxicity present acute tubular necrosis associated with inflammation, which can contribute to intensify the renal damage. Hydrogen sulfide (H2S) is a signaling molecule involved in inflammation. We evaluated the effect of DL-propargylglycine (PAG), an inhibitor of endogenous H2S formation, on the renal damage induced by gentamicin. Male Wistar rats (N = 8) were injected with 40 mg/kg gentamicin (im) twice a day for 9 days, some of them also received PAG (N = 8, 10 mg·kg-1·day-1, ip). Control rats (N = 6) were treated with saline or PAG only (N = 4). Twenty-four-hour urine samples were collected one day after the end of these treatments, blood samples were collected, the animals were sacrificed, and the kidneys were removed for quantification of H2S formation and histological and immunohistochemical studies. Gentamicin-treated rats presented higher sodium and potassium fractional excretion, increased plasma creatinine [4.06 (3.00; 5.87) mg%] and urea levels, a greater number of macrophages/monocytes, and a higher score for tubular interstitial lesions [3.50 (3.00; 4.00)] in the renal cortex. These changes were associated with increased H2S formation in the kidneys from gentamicin-treated rats (230.60 ± 38.62 µg·mg protein-1·h-1) compared to control (21.12 ± 1.63) and PAG (11.44 ± 3.08). Treatment with PAG reduced this increase (171.60 ± 18.34), the disturbances in plasma creatinine levels [2.20 (1.92; 4.60) mg%], macrophage infiltration, and score for tubular interstitial lesions [2.00 (2.00; 3.00)]. However, PAG did not interfere with the increase in fractional sodium excretion provoked by gentamicin. The protective effect of PAG on gentamicin nephrotoxicity was related, at least in part, to decreased H2S formation.

Maturation of endogenous glucose production in preterm and term calves

Glucose disposability is often impaired in neonatal calves and even more in preterm calves. The objective of this study was to investigate ontogenic maturation of endogenous glucose production (eGP) in calves and its effects on postnatal glucose homeostasis. Calves (n = 7 per group) were born preterm (PT; delivered by section 9 d before term) or at term (T; spontaneous vaginal delivery), or spontaneously born and fed colostrum for 4 d (TC). Blood samples were taken immediately after birth and before and 2h after feeding at 24h after birth (PT; T) or on d 4 of life (TC) to determine metabolic and endocrine changes. After birth (PT and T) or on d 3 of life (TC), fasted calves were gavaged with deuterium-labeled water to determine gluconeogenesis (GNG) and intravenously infused with [U(13)C]-glucose to measure eGP and glucose oxidation (GOx) in blood plasma. After slaughter at 26h after birth (PT, T) or on d 4 of life (TC), glycogen concentrations in liver and hepatic mRNA concentrations and enzyme activities of pyruvate carboxylase, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase were measured. Preterm calves had the lowest plasma concentrations of cortisol and 3,5,3'-triiodothyronine at birth. Plasma glucose concentrations from d 1 to 2 decreased more, but plasma concentrations of lactate and urea and glucagon:insulin ratio were higher in PT than in T and TC calves. The eGP, GNG, GOx, as well as hepatic glycogen concentrations and PEPCK activities, were lowest in PT calves. Results indicate impaired glucose homeostasis due to decreased eGP in PT calves and maturation of eGP with ontogenic development.

Intestinal glucose absorption but not endogenous glucose production differs between colostrum- and formula-fed neonatal calves

Glucose supply markedly changes during the transition to extrauterine life. In this study, we investigated diet effects on glucose metabolism in neonatal calves. Calves were fed colostrum (C; n = 7) or milk-based formula (F; n = 7) with similar nutrient content up to d 4 of life. Blood plasma samples were taken daily before feeding and 2 h after feeding on d 4 to measure glucose, lactate, nonesterified fatty acids, protein, urea, insulin, glucagon, and cortisol concentrations. On d 2, additional blood samples were taken to measure glucose first-pass uptake (FPU) and turnover by oral [U-(13)C]-glucose and i.v. [6,6-(2)H(2)]-glucose infusion. On d 3, endogenous glucose production and gluconeogenesis were determined by i.v. [U-(13)C]-glucose and oral deuterated water administration after overnight feed deprivation. Liver tissue was obtained 2 h after feeding on d 4 and glycogen concentration and activities and mRNA abundance of gluconeogenic enzymes were measured. Plasma glucose and protein concentrations and hepatic glycogen concentration were higher (P < 0.05), whereas plasma urea, glucagon, and cortisol (d 2) concentrations as well as hepatic pyruvate carboxylase mRNA level and activity were lower (P < 0.05) in group C than in group F. Orally administered [U-(13)C]-glucose in blood was higher (P < 0.05) but FPU tended to be lower (P < 0.1) in group C than in group F. The improved glucose status in group C resulted from enhanced oral glucose absorption. Metabolic and endocrine changes pointed to elevated amino acid degradation in group F, presumably to provide substrates to meet energy requirements and to compensate for impaired oral glucose uptake.

Islet Neogenesis-associated Protein (ingap): The Role Of Its Endogenous Production As A Positive Modulator Of Insulin Secretion.

Islet neogenesis-associated protein (INGAP) is a peptide found in pancreatic exocrine-, duct- and islet- non-β-cells from normal hamsters. Its increase induced by either its exogenous administration or by the overexpression of its gene enhances β-cell secretory function and increases β-cell mass by a combination of stimulation of cell replication and islet neogenesis and reduction of β-cell apoptosis. We studied the potential modulatory role of endogenous INGAP in insulin secretion using two different experimental approaches. Hamster islets transfected with INGAP-small interfering RNA (INGAP-siRNA) were used to study glucose-stimulated insulin secretion (GSIS). In parallel, freshly isolated islets were incubated with high glucose and the same concentration of either a specific anti-INGAP rabbit serum or normal rabbit serum. INGAP-siRNA transfected islets reduced their INGAP mRNA and protein content by 35.1% and 47.2%, respectively whereas GSIS decreased by 25.8%. GSIS by transfected islets attained levels comparable to those recorded in control islets when INGAP pentadecapeptide (INGAP-PP) was added to the culture medium. INGAP antibody in the medium decreased significantly GSIS in a dose-dependent manner. These results indicate that endogenous INGAP plays a physiological positive modulatory role in insulin secretion, supporting its possible use in the treatment of prediabetes and Type 2 diabetes.

Hypobromous Acid, A Powerful Endogenous Electrophile: Experimental And Theoretical Studies.

Hypobromous acid (HOBr) is an inorganic acid produced by the oxidation of the bromide anion (Br(-)). The blood plasma level of Br(-) is more than 1,000-fold lower than that of chloride anion (Cl(-)). Consequently, the endogenous production of HOBr is also lower compared to hypochlorous acid (HOCl). Nevertheless, there is much evidence of the deleterious effects of HOBr. From these data, we hypothesized that the reactivity of HOBr could be better associated with its electrophilic strength. Our hypothesis was confirmed, since HOBr was significantly more reactive than HOCl when the oxidability of the studied compounds was not relevant. For instance: anisole (HOBr, k2=2.3×10(2)M(-1)s(-1), HOCl non-reactive); dansylglycine (HOBr, k2=7.3×10(6)M(-1)s(-1), HOCl, 5.2×10(2)M(-1)s(-1)); salicylic acid (HOBr, k2=4.0×10(4)M(-1)s(-1), non-reactive); 3-hydroxybenzoic acid (HOBr, k2=5.9×10(4)M(-1)s(-1), HOCl, k2=1.1×10(1)M(-1)s(-1)); uridine (HOBr, k2=1.3×10(3)M(-1)s(-1), HOCl non-reactive). The compounds 4-bromoanisole and 5-bromouridine were identified as the products of the reactions between HOBr and anisole or uridine, respectively, i.e. typical products of electrophilic substitutions. Together, these results show that, rather than an oxidant, HOBr is a powerful electrophilic reactant. This chemical property was theoretically confirmed by measuring the positive Mulliken and ChelpG charges upon bromine and chlorine. In conclusion, the high electrophilicity of HOBr could be behind its well-established deleterious effects. We propose that HOBr is the most powerful endogenous electrophile.

Effect Of Pain Chronification And Chronic Pain On An Endogenous Pain Modulation Circuit In Rats.

We tested the hypothesis that chronic pain development (pain chronification) and ongoing chronic pain (chronic pain) reduce the activity and induce plastic changes in an endogenous analgesia circuit, the ascending nociceptive control. An important mechanism mediating this form of endogenous analgesia, referred to as capsaicin-induced analgesia, is its dependence on nucleus accumbens μ-opioid receptor mechanisms. Therefore, we also investigated whether pain chronification and chronic pain alter the requirement for nucleus accumbens μ-opioid receptor mechanisms in capsaicin-induced analgesia. We used an animal model of pain chronification in which daily subcutaneous prostaglandin E2 (PGE2) injections into the rat's hind paw for 14 days, referred to as the induction period of persistent hyperalgesia, induce a long-lasting state of nociceptor sensitization referred to as the maintenance period of persistent hyperalgesia, that lasts for at least 30 days following the cessation of the PGE2 treatment. The nociceptor hypersensitivity was measured by the shortening of the time interval for the animal to respond to a mechanical stimulation of the hind paw. We found a significant reduction in the duration of capsaicin-induced analgesia during the induction and maintenance period of persistent mechanical hyperalgesia. Intra-accumbens injection of the μ-opioid receptor selective antagonist Cys(2),Tyr(3),Orn(5),Pen(7)amide (CTOP) 10 min before the subcutaneous injection of capsaicin into the rat's fore paw blocked capsaicin-induced analgesia. Taken together, these findings indicate that pain chronification and chronic pain reduce the duration of capsaicin-induced analgesia, without affecting its dependence on nucleus accumbens μ-opioid receptor mechanisms. The attenuation of endogenous analgesia during pain chronification and chronic pain suggests that endogenous pain circuits play an important role in the development and maintenance of chronic pain.

New methods to quantify NH3 volatilization from fertilized surface soil with urea

Gaseous N losses from soil are considerable, resulting mostly from ammonia volatilization linked to agricultural activities such as pasture fertilization. The use of simple and accessible measurement methods of such losses is fundamental in the evaluation of the N cycle in agricultural systems. The purpose of this study was to evaluate quantification methods of NH3 volatilization from fertilized surface soil with urea, with minimal influence on the volatilization processes. The greenhouse experiment was arranged in a completely randomized design with 13 treatments and five replications, with the following treatments: (1) Polyurethane foam (density 20 kg m-3) with phosphoric acid solution absorber (foam absorber), installed 1, 5, 10 and 20 cm above the soil surface; (2) Paper filter with sulfuric acid solution absorber (paper absorber, 1, 5, 10 and 20 cm above the soil surface); (3) Sulfuric acid solution absorber (1, 5 and 10 cm above the soil surface); (4) Semi-open static collector; (5) 15N balance (control). The foam absorber placed 1 cm above the soil surface estimated the real daily rate of loss and accumulated loss of NH3N and proved efficient in capturing NH3 volatized from urea-treated soil. The estimates based on acid absorbers 1, 5 and 10 cm above the soil surface and paper absorbers 1 and 5 cm above the soil surface were only realistic for accumulated N-NH3 losses. Foam absorbers can be indicated to quantify accumulated and daily rates of NH3 volatilization losses similarly to an open static chamber, making calibration equations or correction factors unnecessary.

Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6

Background: Reactivation of p53 by either gene transfer or pharmacologic approaches may compensate for loss of p19Arf or excess mdm2 expression, common events in melanoma and glioma. In our previous work, we constructed the pCLPG retroviral vector where transgene expression is controlled by p53 through a p53-responsive promoter. The use of this vector to introduce p19Arf into tumor cells that harbor p53wt should yield viral expression of p19Arf which, in turn, would activate the endogenous p53 and result in enhanced vector expression and tumor suppression. Since nutlin-3 can activate p53 by blocking its interaction with mdm2, we explored the possibility that the combination of p19Arf gene transfer and nutlin-3 drug treatment may provide an additive benefit in stimulating p53 function. Methods: B16 (mouse melanoma) and C6 (rat glioma) cell lines, which harbor p53wt, were transduced with pCLPGp19 and these were additionally treated with nutlin-3 or the DNA damaging agent, doxorubicin. Viral expression was confirmed by Western, Northern and immunofluorescence assays. p53 function was assessed by reporter gene activity provided by a p53-responsive construct. Alterations in proliferation and viability were measured by colony formation, growth curve, cell cycle and MTT assays. In an animal model, B16 cells were treated with the pCLPGp19 virus and/or drugs before subcutaneous injection in C57BL/6 mice, observation of tumor progression and histopathologic analyses. Results: Here we show that the functional activation of endogenous p53wt in B16 was particularly challenging, but accomplished when combined gene transfer and drug treatments were applied, resulting in increased transactivation by p53, marked cell cycle alteration and reduced viability in culture. In an animal model, B16 cells treated with both p19Arf and nutlin-3 yielded increased necrosis and decreased BrdU marking. In comparison, C6 cells were quite susceptible to either treatment, yet p53 was further activated by the combination of p19Arf and nutlin-3. Conclusions: To the best of our knowledge, this is the first study to apply both p19Arf and nutlin-3 for the stimulation of p53 activity. These results support the notion that a p53 responsive vector may prove to be an interesting gene transfer tool, especially when combined with p53- activating agents, for the treatment of tumors that retain wild-type p53.

Free-standing urethane/urea elastomer films undoped and doped with ferro-nano-particles

We report on an experimental study of the structures presented by urethane/urea elastomeric films without and with ferromagnetic nanoparticles incorporated. The study is made by using the X-ray diffraction, nuclear magnetic resonance (NMR), optical, atomic and magnetic force (MFM) microscopy techniques, and mechanical assays. The structure of the elastomeric matrix is characterized by a distance of 0.46 nm between neighboring molecular segments, almost independent on the stretching applied. The shear casting performed in order to obtain the elastomeric films tends to orient the molecules parallel to the flow direction thus introducing anisotropy in the molecular network which is reflected on the values obtained for the orientational order parameter and its increase for the stretched films. In the case of nanoparticles-doped samples, the structure remains nearly unchanged although the local order parameter is clearly larger for the undoped films. NMR experiments evidence modifications in the molecular network local ordering. Micrometer size clusters were observed by MFM for even small concentration of magnetic particles.

Phytomass production and nitrogen accumulation in maize cultivated with different doses of (15)N-urea

This work aimed to study the possible alterations in production, accumulation of the vegetative phytomass and nitrogen efficiency use of the maize crop, in different doses of N applied in the fertilization, by using the technique of isotopic dilution of (15)N. The completely randomized block experimental design was adopted, with 5 treatments and 4 replicates. The following treatments were constituted in the doses in covering: 0, 50, 100, 150 and 200 kg ha(-1) of N, with fertilization of N-urea, respectively. Comparisons among the treatments had been run for crop productivity; nitrogen accumulation for the plant, and use of the nitrogen of the urea-(15)N for the crop. The increase of the dose of N-fertilizer resulted in increase of the dry matter mass, of the dry matter yield crop tax, of the productivity and accumulation of N in the maize plants.

Influence of Urea and Ammonium Sulfate on Soil Acidity Indices in Lowland Rice Production

Urea and ammonium sulfate are principal nitrogen (N) sources for crop production. Two field experiments were conducted during three consecutive years to evaluate influence of urea and ammonium sulfate application on grain yield, soil pH, calcium (Ca) saturation, magnesium (Mg) saturation, base saturation, aluminum (Al) saturation, and acidity (H + Al) saturation in lowland rice production. Grain yield was significantly influenced by urea as well as ammonium sulfate fertilization. Soil pH linearly decreased with the application of N by ammonium sulfate and urea fertilizers. However, the magnitude of the pH decrease was greater by ammonium sulfate than by urea. The Ca and Mg saturations were decreased at the greater N rates compared to low rates of N by both the fertilizer sources. The Al and acidity saturation increased with increasing N rates by both the fertilizer sources. However, these acidity indices were increased more with the application of ammonium sulfate compared with urea. Rice grain yield had negative associations with pH, Ca saturation, Mg saturation, and base saturation and positive associations with Al and acidity saturation. This indicates that rice plant is tolerant to soil acidity.

Is serum amyloid A an endogenous TLR4 agonist?

Serum amyloid A (SAA), a classical acute-phase protein, is produced predominantly by hepatocytes in response to injury, infection, and inflammation. It has been shown that SAA primes leukocytes and induces the expression and release of proinflammatory cytokines. Here, we report that SAA induces NO production by murine peritoneal macrophages. Using specific inhibitors, we showed that NO production was dependent on inducible NO synthase thorough the activation of ERK1/2 and p38 MAPKs. Moreover, SAA activity was decreased after proteolysis but not with polymyxin B, a lipid A antagonist. Finally, we found that NO production was dependent on functional TLR4, a receptor complex associated with innate immunity. Macrophages from C3H/HeJ and C57BL/10ScCr mice lacking a functional TLR4 did not respond to SAA stimulation. In conclusion, our study makes a novel observation that SAA might be an endogenous agonist for the TLR4 complex on macrophages. The contribution of this finding in amplifying innate immunity during the inflammatory process is discussed.