Renal uptake and excretion of epidermal growth factor from plasma in the rat

The rat excretes around 2 nmol epidermal growth factor (EGF) in the urine per 24 h. The urinary EGF might be derived from plasma and/or might be synthesized in the kidneys. We have used the rat to study the renal uptake and excretion of homologous EGF from plasma. I.v. injected 125I-EGF was removed from the circulation within a few minutes. 5 min after the injection, the kidneys contained 12% of the 125I-EGF. The kidneys seemed to degrade most of the 125I-EGF which they accumulated from blood, as only 4% of the injected label was excreted as intact 125I-EGF in the urine. The amount of endogenous EGF in plasma was under the detection limit of our enzyme-linked immunosorbent assay (0.03 nmol/l) and it remained so after bilateral nephrectomy. Even if plasma EGF was 0.03 nmol/l excretion of EGF from plasma could account for less than 5% of the urinary EGF. This study shows that the kidneys are able to accumulate EGF from plasma and excrete a part of it as intact EGF in the urine. However, excretion of immunoreactive EGF from plasma can only account for a minor part of the urinary EGF.     

The urinary excretion of epidermal growth factor in the rat is reduced by aprotinin, a proteinase inhibitor.

The present study on the rat shows that i.v. administration of the proteinase inhibitor aprotinin reduces the urinary output of immunoreactive epidermal growth factor (EGF) while the amount of immunoreactive EGF in the kidneys is increased. This indicates that the EGF-precursor in the rat kidney in vivo is processed by an aprotinin inhibitable proteinase. EGF is produced in the kidneys as a precursor with a molecular weight of approximately 130 kDa. In rat urine, nanomolar amounts of 6 kDa EGF are excreted per 24 h together with small amounts of high molecular weight forms of EGF. During i.v. administration of aprotinin the median urinary output of immunoreactive EGF is reduced to 15% of the excretion of control rats (23 pmol/2 h versus 157 pmol/2 h, P less than 0.001). Especially the excretion of 6 kDa EGF is reduced (median excretion 12 pmol/2 h versus 134 pmol/2 h, P less than 0.001). The amount of immunoreactive EGF in the kidney tissue is increased after aprotinin administration (median amount 0.11 pmol EGF/mg protein versus less than 0.04 pmol EGF/mg protein, P less than 0.001). Neither the creatinine clearance, the total urinary protein output, nor the volume of urine produced was affected by aprotinin.     

Infusion of epidermal growth factor in mice: organ distribution and urinary excretion

Anesthetized mice were infused into the tail vein with 7.5% mannitol in saline (0.1 ml/min for 60 min) alone or with EGF at 0.5 microgram/min. Urine was collected every 10 min starting 20 min after the beginning of the infusion and ending 20 min after its termination. EGF concentration in the serum of mice infused with EGF increased from the baseline level of 0.6 +/- 0.4 to 70.7 +/- 16.0 ng/ml at 80 min. Total excretion of EGF for 80 min was 117 +/- 49 ng with mannitol alone and 1916 +/- 420 ng (6.4% of the EGF infused) after mannitol with EGF. Serum and urine EGF was indistinguishable from the native mouse EGF by its radioimmunoassay and HPLC characteristics. Intact labeled EGF was also found in urine when mice were infused with 125I-EGF (1 x 10(6) cpm/ml) in mannitol. After 5 min infusion with 125I-EGF (6 x 10(6) cpm/ml in saline), more than 80% of the label was found in the liver and kidneys and more than 90% of it was intact EGF. However, 30 min after infusion more than 95% of the labeled EGF was degraded. We conclude that at least part of the urinary EGF in mice originates in blood and that liver and kidneys are the main organs of EGF degradation.     

Can tritiated water-dilution space accurately predict total body water in chukar partridges?

Total body water (TBW) volumes determined from the dilution space of injected tritiated water have consistently overestimated actual water volumes (determined by desiccation to constant mass) in reptiles and mammals, but results for birds are controversial. We investigated potential errors in both the dilution method and the desiccation method in an attempt to resolve this controversy. Tritiated water dilution yielded an accurate measurement of water mass in vitro. However, in vivo, this method yielded a 4.6% overestimate of the amount of water (3.1% of live body mass) in chukar partridges, apparently largely because of loss of tritium from body water to sites of dissociable hydrogens on body solids. An additional source of overestimation (approximately 2% of body mass) was loss of tritium to the solids in blood samples during distillation of blood to obtain pure water for tritium analysis. Measuring tritium activity in plasma samples avoided this problem but required measurement of, and correction for, the dry matter content in plasma. Desiccation to constant mass by lyophilization or oven-drying also overestimated the amount of water actually in the bodies of chukar partridges by 1.4% of body mass, because these values included water adsorbed onto the outside of feathers. When desiccating defeathered carcasses, oven-drying at 70 degrees C yielded TBW values identical to those obtained from lyophilization, but TBW was overestimated (0.5% of body mass) by drying at 100 degrees C due to loss of organic substances as well as water.(ABSTRACT TRUNCATED AT 250 WORDS)     

A 1H NMR spectroscopic study of the biochemical effects of ifosfamide in the rat: evaluation of potential biomarkers

Ifosfamide (55 mg kg^-1 and 110 mg kg^-1) was administered via single i.p. injections to Sprague-Dawley rats and urine samples were collected for the periods of -24-0, 0-8 h, 8-24 h, 24-48 h and 48-72 h post-dose. Quantitative changes in the excretion pattern of small organic molecules in the urine of rats treated with ifosfamide were studied using high frequency 1H NMR spectroscopy. The kidneys and livers of the animals were also examined, but showed no marked histopathological changes. ¹     

In vitro culture of fibroblast-like cells from postmortem skin of Katahdin sheep stored at 4��C for different time intervals

Live animals have been produced recently from animal tissues preserved for decades at frozen temperatures with or without cryoprotectants. However, the tissues in these studies were cryopreserved within few hours of animal death to obtain culturable live cells as nuclear donors. How long the tissues can be left unfrozen after animal death, without losing the viability and potential to in vitro culture with comparable morphology and proliferative rate as the fresh tissues, is not completely understood. To understand this phenomenon, ear skin samples from individual sheep (n=3) were procured from slaughter plant and stored at 4 °C. After various intervals (2, 8, 24, 32, 48, and 56 h after slaughter), 2-3 mm(2) pieces (n=10) of skin samples were cultured for 12 d on two dishes (60 mm) for each sheep. Outgrowth of fibroblast-like cells was observed as early as day 4 of culture and was visible on dishes of all time points including 56 h by day 10. The number of outgrowing cells decreased with increasing time interval between animal slaughter and culture initiation. Secondary cultures were successfully established for all the time points. All cultures proliferated well and were apparently normal. Passage 2 cultures of 2 h and 56 h interval for one of the three sheep were compared for their growth pattern and proliferation rates. The population doubling time of 2 h and 56 h intervals was 33.12 and 34.8 h, respectively, and both the lines exhibited similar cell morphology and an "S"-shaped growth curve. These results suggest that skin tissues of sheep and perhaps other animal species with superior traits are effectively preserved at cellular level at least for 56 h at normal refrigerating conditions, without need of complicated cryopreservatives/cryotanks that are usually not available at small farms.     

A 1H NMR spectroscopic study of the biochemical effects of ifosfamide in the rat: evaluation of potential biomarkers

Ifosfamide (55 mg kg^-1 and 110 mg kg^-1) was administered via single i.p. injections to Sprague-Dawley rats and urine samples were collected for the periods of -24-0, 0-8 h, 8-24 h, 24-48 h and 48-72 h post-dose. Quantitative changes in the excretion pattern of small organic molecules in the urine of rats treated with ifosfamide were studied using high frequency 1H NMR spectroscopy. The kidneys and livers of the animals were also examined, but showed no marked histopathological changes. ¹     

The source of urinary epidermal growth factor in humans

To clarify the source of human urine EGF, we studied EGF renal clearance in 20 healthy, young adult subjects. Immunoreactive EGF was measured hourly in EDTA plasma, heparin plasma, serum and urine of 12 males and 8 females during a 3 h study period. Plasma and urine creatinine and creatinine clearance were measured and calculated hourly. Mean (and SEM) creatinine clearance was similar in males and females (118 +/- 12 vs 105 +/- 6 ml/min). EGF was not detectable in plasma, whereas relatively high levels were measured in serum (2.5 +/- 0.25 vs 1.5 +/- 0.18 ng/ml in males and females respectively p less than 0.05). Urine EGF excretion averaged 1641 +/- 233 ng/h in males and 1507 +/- 191 ng/h in females (p greater than 0.05). A significant correlation was observed between urine creatinine and urine EGF concentrations in both male (r = 0.98, p less than 0.01) and female (r = 0.94, p less than 0.01) subjects. EGF immunoreactivity in urine and serum eluted from G-75 sephadex columns similarly to recombinant 6000 Mr hEGF. Urine excretion of EGF approximated 1.5 micrograms/h or 25 ng/mg creatine. The high concentrations of EGF found in urine in the face of non-detectable levels of EGF in plasma favor the hypothesis that EGF in urine is derived from kidney synthesis and secretion. The significant positive correlation between urine creatinine and urine EGF suggests a functional correlation between glomerular filtration and the process of tubular EGF excretion.     

Intravenous recombinant secretory leukoprotease inhibitor augments antineutrophil elastase defense.

Secretory leukoprotease inhibitor (SLPI), a 12-kDa serine antiprotease, normally protects the upper airway epithelial surface from attack by neutrophil elastase (NE). In the context that a variety of inflammatory lung diseases are characterized by large neutrophil burdens with resultant high levels of NE in the lung, recombinant SLPI (rSLPI), a molecule identical to natural SLPI, may be an effective means to augment the anti-NE protective screen of the lung. To determine whether intravenous rSLPI will augment respiratory tract and epithelial surface levels of SLPI and anti-NE capacity, rSLPI was administered intravenously to sheep and SLPI levels were quantified in plasma, lung lymph (as a measure of lung interstitial levels), lung epithelial lining fluid (ELF), and urine. rSLPI (1 g) was administered over 10 min, and after 30 min plasma levels of SLPI were 8 microM and decreased with a half-life of 1.8 h. Lymph SLPI levels paralleled the plasma levels: 4 h after infusion the lymph-to-plasma ratio was 0.8. ELF SLPI levels paralleled the lymph levels: 4 h after infusion the ELF-to-lymph ratio was 0.3. Western analysis demonstrated intact SLPI in lymph and ELF, and functional analysis showed increases in lymph and ELF anti-NE capacity that paralleled the levels of SLPI. As might be expected from a protein with a molecular mass of 12 kDa, urine excretion was high, with 20% of the SLPI excreted over 5 h. However, if the rate of infusion was slowed, SLPI excretion decreased significantly, with a 3-h infusion associated with 9% excretion and a 12-h infusion associated with less than 0.2% excretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of organ uptake and disappearance half-time of human epidermal growth factor and insulin

Epidermal growth factor (EGF), which was originally identified in salivary glands and saliva, has been also found in the kidney and urine, suggesting that the kidney may be an alternate source of this peptide. Liver was considered as the major site of the degradation of EGF but the involvement of other organs has been little studied. Therefore, we carried out comparative studies on the organ uptake and the disappearance half-time of EGF and insulin (having similar molecular size) in the same model of anesthetized dog with arterial (from aorta) and venous (from mesenteric, portal, hepatic, renal, femoral and jugular veins) blood sampling from various organs. Basal plasma level of EGF (1.32 +/- 0.33 pmol/l) and insulin (62.1 +/- 13.8 pmol/l) in the aorta was not significantly different from that recorded at various sampling sites. During i.v. infusion of EGF at 41.6 and 166.6 pmol/kg/h, the respective arterial EGF concentrations averaged 103 +/- 21 and 240 +/- 49 pmol/kg/h and the percent reduction in plasma EGF after passage through the head, leg, intestines and liver was about 30-50% and that after passage through the kidney was about 95%. During insulin (6.9 pmol/kg/h) infusion, the arterial hormone level averaged 227 +/- 21 pmol/l and this level was significantly reduced (by 23-42%) after passage through the head, leg, intestine, liver and kidney but no significant difference was found between various venous sampling sites. EGF and insulin appearing in the urine during EGF or insulin infusion accounted for about 40 and 7% of the difference between the entering and leaving renal masses of the peptide. Mean disappearance half time on stopping of EGF and insulin infusion was, respectively, 2.32 +/- 0.58 and 6.88 +/- 1.25 min. We conclude that unlike insulin, which is removed to similar extent by various organs including the kidney and the liver, EGF is taken up mainly by kidney and EGF present in urine originates mainly from renal clearance of peptide.     

Determination by ICP-MS and multivariate data analysis of elemental urine excretion profile during the EDTA chelation therapy: A case study

BackgroundBased on the medical history and laboratory analytical tests, a patient presenting symptoms compatible with Chronic Fatigue Syndrome was suspected of metal intoxication; therefore, a chelating therapy was attempted. In parallel, the profile of elemental excretion in urine was determined.MethodsChelation therapy by CaNa₂EDTA was administered every two weeks and urine samples were routinely collected for 17 months. The samples were mineralized with HNO₃ 69 % and analyzed by Inductively-Coupled Plasma – Mass Spectrometry. Data were processed by multivariate statistical methods.ResultsMost of the toxic elements showed a peak of excretion in 12−24 h after EDTA administration, which returned to basal level by 24−36 h after the treatment. Yet, the excretion of some trace elements persisted in the urine collected 26 h after the treatment.ConclusionsThe analysis of excreted metals following the CaNa₂EDTA infusion allowed to monitor dynamically the chelation therapy. The chelation therapy was effective in mobilizing and eliminating the principal heavy metals present from the body. However, since such clearance almost vanished 24 h after the treatment, a protocol with more frequent and low-dose administrations is advisable to improve the metal excretion.     

Gas chromatographic determination of inorganic tin in rat urine after a single oral administration of stannous chloride and mono-, di-, and triphenyltin chloride

A method is described for the determination of inorganic tin by gas chromatography with flame photometric detection. The inorganic tins, stannous and stannic, were extracted with hydrochloric acid and n-hexane—benzene in the presence of 0.05% tropolone, and both inorganic tins were pentylated to tetrapentyltin with a Grignard reagent prior to gas chromatography. The absolute limit of detection for tetrapentyltin was 3 pg as tin. The recovery of stannous chloride added to rat urine samples was 80.2 ± 2.4% (mean ± S.D., n = 8). The application of this method to the study of urinary excretion of inorganic tin and organotin compounds in rats following oral administration of tin compounds is presented. The urinary excretion of tin compounds was observed over a period of 96 h following administration of stannous chloride or phenyltin compounds. Most of the inorganic tin was excreted into urine within 24 h after administration of stannous chloride. In the experiments on organotin administration, the level of the excretion as total tin for monophenyltin reached a maximum ca. 0–24 h after administration, whereas the maxima for di- and triphenyltin were found after 24–48 h and 48–72 h, respectively. The predominant excretion product of these tin compounds found in urine was monophenyltin.     

Effects of acute intravenous aldosterone administration on Na(+), K(+), and water excretion in the horse.

The effect of a temporary increase in plasma aldosterone concentration on Na(+), K(+), and water balance was investigated in four horses. Aldosterone was injected intravenously for 6 h at 20-min intervals (total 5.4 microg/kg body wt). Samples were taken for 24 h before, during, and for 48 h after the treatment. Aldosterone treatment reduced the Na(+) loss via urine and feces by 99 and 72%, respectively, later followed by a marked increase in Na(+) excretion by both pathways. During the first 6 h after the treatment, fecal K(+) excretion was elevated, and the plasma K(+) concentration was lowered. Fluid was retained throughout the treatment period and for 12-15 h thereafter. In a second experiment, exercise was performed once after aldosterone treatment and once without prior treatment. Sweat samples were collected, and the composition was not altered after treatment. It was concluded that acute aldosterone injections reduce Na(+) losses in both feces and urine but not in sweat. In addition, the feces was shown to be the main excretion pathway of aldosterone.     

Feed deprivation in Merino and Terminal sired lambs: (2) the metabolic response under pre-slaughter conditions and impact on meat quality and carcass yield

Under current Australian industry pre-slaughter guidelines, lambs may be off feed for up to 48 h before slaughter. The purpose of this study was to examine what proportion of circulating metabolites at slaughter are due to stress and feed deprivation and if this response differs between Merino and Terminal genotypes. In addition the effect of feed deprivation on carcass weight and meat quality was examined. Jugular blood samples were collected from 88 Merino and Terminal sired lambs at rest and at slaughter following 24, 36 and 48 h of feed deprivation and plasma analysed for glucose, lactate, non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHOB). From the same carcasses hot carcass weight (HCWT) were measured as well as a suite of meat quality traits measured such as M. longissimus lumborum (loin) and M. semitendinosus pH at 24 h postmortem. Loin samples were also analysed for intramuscular fat content and Warner–Bratzer Shear Force. Merino sired lambs had a higher NEFA response compared to Terminal sired lambs at slaughter after 24, 36 and 48 h of feed deprivation, with NEFA levels up to 35% higher than previously reported in the same animals at rest in animal house conditions, whereas BHOB response to feed deprivation was not affected by sire type (P>0.05) and similar to previously reported at rest. In addition to the metabolic effects, increasing feed deprivation from 36 h was associated with a 3% reduction in HCWT and dressing percentage as well as causing increased ultimate pH in the M. semitendinosus in Merino sired lambs. Findings from this study demonstrate that Merino and Terminal sired lambs differ in their metabolic response to feed deprivation under commercial slaughter conditions. In addition, commercial feed deprivation appears to have a negative effect on ultimate pH and carcass weight and warrants further investigation.     

Mutagenicity induced by lyophilization or storage of urine from isoniazid-treated rats.

Urine collected during 24 h after treatment of rats with 90--550 mg/kg isonicotinic acid hydrazide (isoniazid, INH) was after lyophilization, mutagenic for Salmonella typhimurium TA1535. Urine collected directly from bladders of INH-treated rats was not mutagenic, and solutions of INH in water or urine became mutagenic only after lyophilization. In the absence of lyophilization, sterile urine from INH-treated rats became mutagenic after 8--14 days' storage at room temperature.     

Epidermal growth factor alters the electrolyte profile of lactating ewes Ovis aries

• 1.1. Lactating ewes were treated with mouse epidermal growth factor (EGF) at a dose rate of 0.5 mg/day for 4 days and its effects on the electrolyte profile were observed.
• 2.2. There was no effect of EGF on plasma concentrations of sodium or potassium, although urinary and total (in urine and milk) losses of both were reduced.
• 3.3. EGF-induced hypocalcaemia was associated with reduced milk calcium secretion and increased urinary calcium excretion whereas EGF-induced hypermagnesaemia was associated with reduced urinary and total magnesium losses.
• 4.4. Glomerular filtration rate was reduced during EGF infusion.
• 5.5. Chronic intravenous EGF infusion affects the electrolyte profile by altering electrolyte secretion by the mammary gland and renal electrolyte excretion.

     

Fate of mimosine administered orally to sheep and its effectiveness as a defleecing agent.

Mimosine was administered orally to Merino sheep once daily for periods of 1-3 days, either as the isolated compound or in the foliage of Leucaena leucocephala. A single daily dose of mimosine of 450 or 600 mg/kg body weight was effective for defleecing sheep. A daily dose rate of 300 mg/kg was effective for defleecing sheep if given on two successive days. The effectiveness of a treatment for defleecing sheep was related to the concentration of mimosine in plasma following dosing; defleecing ensued when the concentration of mimosine in plasma was maintained above 0-1 mmol/l for at least 30 h. The main products excreted in urine were mimosine and 3,4-dihydroxypyridine (DHP); small amounts of mimosinamine were also excreted. During the first day following dosing, the major excretory product was mimosine; DHP was an important component during the second and third days. In the three days following the start of dosing, between 32 and 53% of the mimosine given was accounted for as mimosine in the urine. Following an intravenous infusion of mimosine, no DHP was detected in urine; most of the mimosine was excreted intact but a small amount (c. 9%) was excreted as mimosinamine.     

Systems for collection of urine in the captive common marmoset, Callithrix jacchus

Two systems are described for the collection of 24 h urine samples from the common marmoset (Callithrix jacchus). Using 84 adult animals, 1210 24-h samples were collected. Mean urinary excretion was 14.4 +/- 7.5 ml/24 h (n = 1210, mean +/- SD). No differences were observed between sexes (for 52 females, 24 h volume = 15.1 +/- 8.0 ml; for 32 males, 24 h volume = 12.5 +/- 6.0 ml). No significant differences were observed between pregnant and non-pregnant females with respect to 24 h urine volume, and bilateral gonadectomy did not influence subsequent urinary excretion in either sex. For 161 pairs of observations, the intake of drinking water (11.7 +/- 10.2 ml/24 h) and the volume of urine excreted (12.6 +/- 7.1 ml/24 h) showed a positive correlation (r = 0.406 d.f. 159, P less than 0.001: y = 0.558x + 4.247).     

Exposure of bovine oocytes to EGF during maturation allows them to develop to blastocysts in a chemically-defined medium

When cumulus-enclosed bovine oocytes were cultured for 24 h in serum-free medium containing 0 to 50 ng/ml EGF, the proportions of oocytes reaching metaphase II were higher (P < 0.05) in the presence of 30 ng/ml EGF (88.1 +/- 1.3%) than under control conditions (65.5 +/- 3.5%) or in the presence of 10 ng/ml (73.9 +/- 4.5%) and 50 ng/ml (73.6 +/- 4.0%) EGF. When oocytes matured under these conditions were inseminated in vitro, the proportions of oocytes penetrated were higher (P < 0.05) in 10 to 50 ng/ml EGF (96.7 +/- 3.3 to 100%) than in its absence (77.9 +/- 8.9%). However, the proportions of penetrated oocytes with male and female pronuclei did not differ among the different groups (96.7 +/- 3.3 to 100%). When oocytes were matured under the same conditions, fertilized in vitro, and cultured until 192 h post insemination in a chemically-defined medium, the proportion of embryos at the >/=2-cell stage was higher (P < 0.05) in the groups treated with 30 ng/ml (96.1 +/- 2.5%) and 50 ng/ml (90.6 +/- 3.5%) EGF than in the controls (71.8 +/- 3.1%) at 48 h post insemination. Although there were no differences in the proportions (37.3 +/- 5.3 to 47.2 +/- 5.8%) of >/=morulae at 144 h post insemination among treatments, the proportion of embryos developing to the blastocyst stage was higher (P < 0.05) in the presence of 10 to 50 ng/ml EGF (16.5 +/- 2.0 to 20.8 +/- 4.9%) than in control medium (3.4 +/- 2.1%). The mean blastocyst cell number at 192 h post insemination did not differ between culture media in the presence (91 to 107 cells) and the absence (116 cells) of EGF (10 to 50 ng/ml) during maturation. Thus, higher proportions of oocytes matured in serum-free medium with EGF than without EGF could develop to the blastocyst stage in a chemically-defined medium after in vitro fertilization. These results indicate that EGF can induce not only nuclear maturation but also cytoplasmic maturation of cumulus-enclosed bovine oocytes in vitro.     

Cellular proliferation during cicatrization of the skin of 7-day chick embryos cultured in vitro

A cell proliferation study during in vitro wound healing of dorsal thoraco-lumbar skin of 7-day chick embryos was performed by pulse labelling using a single isotope (tritiated thymidine). The unoperated (controls) and operated explants were incubated in the radioactive medium (1 microCi/ml tritiated thymidine) 1 h prior to fixation and where fixed 1 h (start control), 48, 72 and 96 h after the excision. Mean labelling and mitotic indices of the unwounded epidermis were, respectively, 18.22% and 2.66% at 7 days, and 7.03% and 0.88% at 11 days (7 days + 96 h). 72 h after the excision, the labelling and mitotic indices of wounded epidermis increased, on average, respectively to 212,5% and 220% with respect to those of the controls, in the proximal zones near the inner edge on the wound. The labelling and mitotic indices in the dermis were, respectively, 27.95% and 3.63% at 7 days and 7.65% and 1.30% at 11 days. 72 h after the excision, the labelling and mitotic indices of the operated dermis increased, on average, respectively to 220% and 130% with respect to those of the controls in the centre and the proximal zones of the wound. The increase of the labelling index of the operated integument persisted for a maximum of 24 h, between 48 to 72 hours after the excision.