The effects of sepsis and endotoxemia on gut glutamine metabolism.

The effects of sepsis on gut glutamine (GLN) metabolism were studied to gain further insight into the regulation of the altered glutamine metabolism that characterizes critical illnesses. Studies were done in laboratory rats and in hospitalized patients. The human studies were done in seven healthy surgical patients (controls) and six septic patients who underwent laparotomy. Radial artery and portal vein samples were obtained during operation and were analyzed for GLN and oxygen content. Despite no reduction in arterial glutamine concentration in the septic patients, gut glutamine extraction was diminished by 75% (12.0% +/- 1.6% in controls vs. 2.8% +/- 0.8% in septic patients, p less than 0.01). Similarly gut oxygen extraction was diminished by nearly 50% in the septic patients (p less than 0.05). To further investigate these abnormalities, endotoxin (10 mg/kg intraperitoneally) or saline (controls) was administered to adult rats 12 hours before cannulation of the carotid artery and portal vein. The arterial GLN concentration was increased by 13% in the endotoxin-treated animals (p less than 0.05) but gut glutamine uptake was diminished by 46% (526 +/- 82 nmol/100 g BW/minute in controls vs. 282 +/- 45 in endotoxin, p less than 0.01). Simultaneously gut glutaminase activity was diminished by 30% (p less than 0.01) and intestinal glutamate release fell by two thirds. Blood cultures were negative in control animals (0 of 20), but were positive in 25% of endotoxemic animals (6 of 24) for gram-negative rods (p = 0.019). Sepsis and endotoxemia impair gut glutamine metabolism. This impairment may be etiologic in the breakdown of the gut mucosal barrier and in the development of bacterial translocation.     

Glutamine metabolism by the endotoxin-injured lung.

The alterations in lung glutamine (GLN) metabolism that occurs in the endotoxin-injured lung were studied in rats and subsequently correlated with flux changes that occur in patients with the adult respiratory distress syndrome (ARDS). Measurements in animals were made at various time-points following the administration of endotoxin, while studies in surgical patients were done in a group of healthy controls, in patients with "early" sepsis who had normal chest x-ray films, and in patients with radiographic and physiologic evidence of ARDS. In healthy control rats, net amounts of GLN are released by the lungs into the systemic circulation. This release rate doubled 30 minutes after intravenous endotoxin (1,580 +/- 320 nmol GLN/100 g BW/min vs. 736 +/- 179 in controls, p less than 0.01) but glutamine synthetase activity was unchanged, suggesting an outpouring of cellular glutamine stores. Two hours after endotoxin treatment, this accelerated fractional release of glutamine by the lungs was no longer detected. By the 12-hour time-point, the lungs reversed to an organ of net glutamine balance (234 +/- 248 nmol/100 g BW/min, p less than 0.05 vs. controls and ENDO30 min) despite a more than two-fold increase in glutamine synthetase activity (p less than 0.01). Simultaneously, lung weights were increased by 21% (p less than 0.01) and histologic examination showed an interstitial infiltrate and pulmonary edema. Similar observations were made in humans; patients with "early" sepsis exhibited a marked increase in lung glutamine release, while patients with ARDS demonstrated glutamine balance across the lungs (4,030 +/- 910 nmol GLN/kg BW/min vs. 637 +/- 496 in ARDS, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytokine regulation of intestinal glutamine utilization.

The effects of cytokines on intestinal glutamine metabolism were studied to gain further insight into the regulation of altered glutamine metabolism that occurs during severe infection. One hundred thirteen adult rats were given a single dose of interleukin-1 (IL-1, 50 micrograms/kg), tumor necrosis factor (TNF, 50 micrograms/kg or 150 micrograms/kg), or saline (controls), and flux studies were performed 4 or 12 hours later. Intestinal blood flow was not different between control and cytokine-treated animals at either time point. At the 4-hour time point, arterial glutamine fell by 16% to 21% in the cytokine-treated animals (p less than 0.05); at the 12-hour time point, the arterial glutamine concentration had returned to normal. Intestinal glutamine extraction decreased in the animals treated with IL-1 at both time points (4 hours: 13% +/- 1.3% in IL-1 versus 20% +/- 1.6% in controls, p less than 0.05; and 12 hours: 9% +/- 2% in IL-1 versus 17% +/- 2% in controls, p less than 0.05). Consequently, net intestinal glutamine uptake fell in the animals treated with IL-1 at both time points (p less than 0.05). Similarly, the activity of mucosal glutaminase, the principal enzyme of glutamine hydrolysis in the gut, fell by 50% in the 4-hour study (6.1 +/- 0.6 mumol/h/mg protein in IL-1 versus 9.6 +/- 0.8 mumol/h/mg protein in controls, p less than 0.01) and by 40% in the 12-hour study (5.4 +/- 0.5 mumol/h/mg protein in IL-1 versus 8.8 +/- 0.4 mumol/h/mg protein in controls, p less than 0.05). Concomitant with the aforementioned decrease in gut glutamine metabolism was a 25% incidence of positive blood cultures for gram-negative organisms in IL-1 treated rats studied at the 12-hour time point (p = 0.05 versus controls). In the doses administered and at the time points studied, TNF had no effects on the parameters of gut glutamine metabolism examined. The results indicate that IL-1 is a potential mediator of the alterations in gut glutamine metabolism observed in sepsis and endotoxemia.     

The effects of endotoxin on glutamine transport by pulmonary artery endothelial cells

The effects of endotoxin on glutamine transport by cultured pulmonary artery endothelial cells (PAECs) were studied in order to gain further insight into the regulation of the altered lung glutamine metabolism that characterizes severe infection. Incubation of PAECs with endotoxin (1 micrograms/ml) resulted in a significant increase in System ASC-mediated glutamine transport which did not occur for 8 hr and was maximal after 12 hr of exposure. Kinetic studies indicated that the increase in carrier-mediated activity was not due to a change in Km (101 +/- 6 microM in controls vs 97 +/- 4 microM in endotoxin-treated cells, P = NS), but rather to a 73% increase in Vmax (840 +/- 60 pmole/mg protein/30 sec in controls vs 1450 +/- 80 in endotoxin-treated cells, P less than 0.001). The increase in glutamine uptake by PAECs was completely blocked by actinomycin D and cycloheximide, indicating that the accelerated glutamine transport was most probably due to an increase in transporter synthesis. Endotoxin stimulates glutamine uptake by PAECs, either directly or indirectly, an adaptive response which may be necessary to support cellular metabolism, structure, and function.     

Effect of supplemental dietary glutamine on methotrexate concentrations in tumors.

This study evaluated the effects of supplemental dietary glutamine (GLN) on methotrexate sodium concentrations in tumors and serum of sarcoma-bearing rats following the initiation of methotrexate. After randomization to a GLN diet (+GLN) or GLN-free diet (-GLN), tumor-bearing rats received 20 mg/kg of methotrexate sodium by intraperitoneal injection. The provision of supplemental GLN in the diet increased methotrexate concentrations in tumor tissues at 24 and 48 hours (38.0 +/- 0.20 nmol/g for the +GLN group vs 28.8 +/- 0.10 nmol/g for the -GLN group and 35.6 +/- 0.18 nmol/g for the +GLN group vs 32.5 +/- 0.16 nmol/g for the -GLN group, respectively). Arterial methotrexate levels were elevated only at 48 hours (0.147 +/- 0.007 microns/L for the +GLN group vs 0.120 +/- 0.006 microns/L for the -GLN group). Tumor morphometrics were not different between the groups but significantly greater tumor volume loss was seen even at 24 hours (-2.41 +/- 1.3 cm3 for the +GLN group vs -0.016 +/- 0.9 cm3 for the -GLN group). Tumor glutaminase activity was suppressed in both groups at 48 hours, but more so in the +GLN group (0.94 +/- 0.13 mumol/g per hour for the +GLN group vs 1.47 +/- 0.22 mumol/g per hour for the -GLN group). This study suggests that GLN may have therapeutic as well as nutritional benefit in oncology patients.     

The early response of the jejunal brush border glutamine transporter to endotoxemia.

The early effects of endotoxin (4 hr after a single dose of Escherichia coli LPS, 7.5 mg/kg) on L-glutamine (GLN) transport across the jejunal brush border of rats were studied. Jejunal brush border membrane vesicles (BBMVs) were prepared by a Mg2+ aggregation/differential centrifugation technique. Vesicle purity and integrity were confirmed by a 15-fold enrichment of brush border marker enzymes, osmotic activity, transport overshoots in the presence of sodium, and similar 1- and 2-hr equilibrium values. L-[3H]GLN transport in jejunal BBMVs was measured by a millipore filtration technique. Na(+)-dependent glutamine transport, which accounted for greater than 80% of total transport, was increased twofold in BBMVs from endotoxin-treated rats (67 +/- 5 pmole/mg protein/15 sec vs 38 +/- 3, P less than 0.01). Endotoxin treatment did not alter the activity of the Na(+)-independent carrier. Simultaneously, intestinal extraction of glutamine from the bloodstream fell by 56% (15.1 +/- 2.3% in controls vs 6.6 +/- 1.3% in endotoxin-treated rats, P less than 0.01). This reduction in the uptake of circulating glutamine could not be accounted for by a fall in the arterial concentration. Thus, soon after endotoxemia brush border glutamine uptake is increased while consumption of glutamine across the basolateral membrane is decreased. This increased uptake may support protein synthesis and may provide a biochemical rationale for the use of early enteral nutrition after the onset of critical illness.     

Adaptive regulation in skeletal muscle glutamine metabolism in endotoxin-treated rats.

The effects of a single dose of endotoxin (7.5 mg/kg BW) on skeletal muscle glutamine metabolism were studied in vivo in rats to gain further understanding of the altered glutamine metabolism that characterizes sepsis and other catabolic diseases. In endotoxin-treated animals the arterial glutamine concentration fell early initially and then increased compared with control values. Twelve hours after treatment, the arteriovenous concentration difference for glutamine across the hindquarter doubled, resulting in a significant increase in net muscle glutamine release in endotoxin-treated rats. As a consequence, the muscle glutamine concentration fell in the endotoxin-treated animals by 25%-40%, an event that was apparent as early as two hours after endotoxin treatment. Skeletal muscle glutaminase activity, the major enzyme of glutamine breakdown, was unchanged by endotoxemia, but expression of glutamine synthetase mRNA and glutamine synthetase specific activity increased in a time-dependent fashion. The glutamine depletion that develops in skeletal muscle during endotoxemia is caused by accelerated muscle glutamine release rather than an increase in intracellular degradation or a fall in intracellular biosynthesis. The adaptive increase in glutamine synthetase expression that occurs requires de novo RNA and protein synthesis and may be designed to prevent complete depletion of the intracellular glutamine pool.     

Brush border transport of glutamine and other substrates during sepsis and endotoxemia

The effects of severe infection on luminal transport of amino acids and glucose by the small intestine were investigated. Studies were done in endotoxin-treated rats and in septic patients who underwent resection of otherwise normal small bowel. In rats the kinetics of the brush border glutamine transporter and the glutaminase enzyme were examined. In patients the effects of severe infection on the transport of glutamine, alanine, leucine, and glucose were studied. Transport was measured using small intestinal brush border membrane vesicles that were prepared by Mg++ aggregation/differential centrifugation. Uptake of radiolabeled substrate was measured using a rapid mixing/filtration technique. Vesicles demonstrated 15-fold enrichments of enzyme markers, classic overshoots, transport into an osmotically active space, and similar 2-hour equilibrium values. The sodium-dependent pathway accounted for nearly 90% of total carrier-mediated transport. Kinetic studies on rat jejunal glutaminase indicated a decrease in activity as early as 2 hours after endotoxin secondary to a decrease in enzyme affinity for glutamine (Km = 2.23 +/- 0.20 mmol/L [millimolar] in controls versus 4.55 +/- 0.67 in endotoxin, p less than 0.03), rather than a change in Vmax. By 12 hours the decrease in glutaminase activity was due to a decrease in Vmax (222 +/- 36 nmol/mg protein/min in controls versus 96 +/- 16 in endotoxin, p less than 0.03) rather than a significant change in Km. Transport data indicated a decrease in sodium-dependent jejunal glutamine uptake 12 hours after endotoxin secondary to a 35% reduction in maximal transport velocity (Vmax = 325 +/- 12 pmol/mg protein/10 sec in controls versus 214 +/- 8 in endotoxin, p less than 0.0001) with no change in Km (carrier affinity). Sodium-dependent glutamine transport was also decreased in septic patients, both in the jejunum (Vmax for control jejunum = 786 +/- 96 pmol/mg protein/10 sec versus 417 +/- 43 for septic jejunum, p less than 0.01) and in the ileum (Vmax of control ileum = 1126 +/- 66 pmol/mg protein/10 sec versus 415 +/- 24 in septic ileum, p less than 0.001) The rate of jejunal transport of alanine, leucine, and glucose was also decreased in septic patients by 30% to 50% (p less than 0.01). These data suggest that there is a generalized down-regulation of sodium-dependent carrier-mediated substrate transport across the brush border during severe infection, which probably occurs secondary to a decrease in transporter synthesis or an increase in the rate of carrier degradation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Renal arteriovenous fistula: a model for renovascular hypertension in rat

Renal arteriovenous fistula (RAVF) was created in the rat. For this purpose the left renal artery and vein were connected by a latero lateral anastomosis. This operation was performed in 40 rats and 12 animals were sham operated. All these animals were subsequently reoperated after 4 weeks to check the fistula and the kidney. Eleven among the 40 operated rats died during or after the surgery. In 16 others animals hypertension developed. Their blood pressure was 161 +/- 4.3 mm. Hg at 4 weeks vs. 105 +/- 1.65 mm. Hg before surgery. All these animals had after 4 weeks a permeable fistula and an atrophic but nonnecrotized left kidney (kidney weight was 0.56 +/- 0.33 gm. vs. 1.32 +/- 0.22 gm. for the sham operated group). They underwent a left nephrectomy without closure of the fistula and subsequently their blood pressure fell back to normal values 120 +/- 2.9 mm. Hg 2 weeks after nephrectomy). In the removed kidney, renal renin content per mg. of protein was found to be higher than in the kidney of the sham operated animals (28.5 +/- 7.9 vs. 0.66 +/- 0.2 micrograms. AI/mg. protein/hour). In 13 other animals the blood pressure remained normal after the initial surgery. These animals had a permeable fistula but a necrotic left kidney. It is concluded that RAVF can induce hypertension in the rat. This hypertension only develops when the ipsilateral kidney remains vascularized and disappears when this kidney is removed without closure of the fistula. Since the renin content in the kidney distal to the shunt was higher than in the kidney of the sham operated rats and since there was a positive correlation between renal renin content and blood pressure in hypertensive animals, it is suggested that an activation of the renin angiotensin system in the ipsilateral ischemic kidney is responsible for hypertension. RAVF therefore appears to be an experimental model for renovascular hypertension in rats.     

Failure of verapamil to protect from ischaemic renal damage

To reassess the reported protective effects of verapamil in renal ischaemia, we perfused the left kidney of uninephrectomized female Wistar rats with verapamil (0.1 and 1.0 mg/kg) immediately prior to clamping the renal artery for 60 min. When compared to controls, both doses of verapamil failed to afford protection with respect to urine flow, plasma creatinine, creatinine clearance or histology 24 and 48 h after ischaemia, whereas the purine nucleotide inosine (200 mg/kg) was partially protective: mean plasma creatinine 48 h after ischaemia (+/- SEM) was 547 +/- 54 mumol/l in controls, 686 +/- 38 mumol/l with 0.1 mg/kg verapamil, 491 +/- 68 mumol/l with 1.0 mg/kg verapamil and 374 +/- 45 mumol/l with inosine (p less than 0.05 vs. controls). Using the same model, the effect of verapamil 1.0 mg/kg on renal blood flow, creatinine clearance, urine flow and arterial pressure was studied in the first 2 h after ischaemia. Although significant amounts of verapamil were present in the kidney during ischaemia as evidenced by decreases in systemic blood pressure and in renal vascular resistance after unclamping the renal artery, the early course of renal failure was not altered when compared to controls. In conclusion, we have been unable to confirm earlier reports of a protective effect of verapamil in this rat model of ischaemic renal failure. If there is such an effect, its demonstration appears to depend on very specific experimental circumstances. Based on the results of this and other studies, verapamil is unlikely to afford an impressive overall benefit in the clinical setting of ischaemic renal failure.     

The effect of calcitonin-gene-related peptide on acute ischemia-reperfusion renal injury: ultrastructural and membrane lipid peroxidation studies.

It is well accepted that postischemic reperfusion promotes functional and morphological impairment which may be related to oxygen free-radical-mediated membrane damage. A new purified bioactive compound, calcitonin-gene-related peptide (CGRP), is known to be not only a potent vasodilator but also a cytoprotective agent. This study was designed to observe whether CGRP has a protective effect on the ischemic kidney. Male Sprague-Dawley rats were subjected to a 45-min period of renal ischemia followed by 60 min of reperfusion. At the beginning of the reperfusion, 12 rats were given intravenous saline and served as controls whereas 5 rats were given CGRP, 10 micrograms/kg intravenously. After reperfusion the kidneys were removed for light- and electronmicroscopy, and the lipid peroxidation product malonaldehyde (MDA) was assayed by thiobarbituric acid (TBA) colorimetry. The results demonstrated that the serum creatinine (Scr) and renal MDA content in the CGRP group were significantly lower than those in the control group. The mean values for Scr were 0.75 +/- 0.09 vs 0.93 +/- 0.05 mg/dL or 62.8 +/- 9.7 vs 82.2 +/- 4.4 mumol/L (p less than 0.05), respectively; while the mean values for MDA were 18.71 +/- 2.13 vs 30.32 +/- 1.78 nmol/100 mg (ww) (p greater than 0.05), respectively. The same signals of free radicals in the ischemic-reperfused kidney with or without CGRP were found by electron spin resonance. Morphological studies demonstrated that the treatment with CGRP ameliorated the ischemic-reperfusion injury to both renal brush borders and mitochondria. The results showed that CGRP has a protective action on ischemia-reperfusion renal injury by decreasing lipid peroxidation of membranes and suggest that it may be a beneficial agent for therapy of acute renal failure.     

Remnant kidney metabolism in the dog.

A marked increase in oxygen uptake (Qo2) per nephron has been described in the remnant kidney of the rat. However, it is not known which substrates support renal metabolism in remnant kidney nor is it known whether similar changes in Qo2 occur in other species. Remnant kidney in the dog was induced by ligation of 60 to 75% of the renal arterial branches on one side followed 1 to 2 wk later by contralateral nephrectomy. At 3 months marked hypertrophy of the remnant kidney was found and the glomerular filtration rate was 18 +/- 1.8 mL/min compared with 31 +/- 2 in a normal kidney (P less than 0.01). Qo2 was 689 +/- 60 mumol/min/100 mL glomerular filtration rate in the remnant kidney compared with 564 +/- 42 mumol/min/100 mL glomerular filtration rate in the normal kidney (P less than 0.01). Total renal ammoniagenesis per nephron increased to values found in chronic metabolic acidosis although serum (K+) and (HCO3-) were no different than in the normal dog. The oxidation of glutamine and lactate by remnant kidneys accounted for over 80% of Qo2, similar to that of normal kidneys. It is concluded that hypermetabolism per nephron occurs in the remnant kidney of the dog and that glutamine and lactate are the major energy substrates in remnant kidney. Furthermore, factors other than serum (K+) and (HCO3-) augment ammoniagenesis in this model. However, when these results are expressed per whole kidney or per gram of tissue, hypermetabolism does not occur in these remnant kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)     

Melatonin, a pineal hormone with antioxidant property, protects against gentamicin-induced nephrotoxicity in rats

The present study investigated the effects of melatonin, an antioxidant, on gentamicin-induced nephrotoxicity in rats. Melatonin (5 mg/kg p.o.) was used 3 days before and 8 days simultaneously with gentamicin (80 mg/kg i.p.) Saline-treated animals served as controls. Determinations of urinary creatinine, N-acetyl-beta-D-glucosaminidase, glucose, protein, blood urea, serum creatinine, plasma and kidney tissue malondialdehyde (MDA), and antioxidant enzyme levels in kidney tissue were done after 8 days of gentamicin treatment. The kidneys were also examined for morphological changes using histological techniques. Gentamicin caused nephrotoxicity as evidenced by marked elevation in blood urea and serum creatinine. Mean blood urea and serum creatinine levels were 289+/-50, and 2.5+/-0.5 mg/dl, respectively, in rats treated with gentamicin. Melatonin significantly protected the rats from gentamicin-induced nephrotoxicity; blood urea and serum creatinine levels were 23+/-2.7 and 0.88+/-0.19 mg/dl, respectively. The creatinine clearance was decreased with gentamicin treatment (0.048+/- 0.007 ml/min) as compared with controls (0.41+/-0.08 ml/h/kg). In rats treated with melatonin plus gentamicin, the creatinine clearance was similar to controls (0.41+/-0.08 ml/h/kg). The product of lipid peroxidation (MDA) was markedly increased in plasma (2.10+/-0.15 nmol) and kidney tissue (8.87+/-3.2 nmol/mg protein) with gentamicin treatment. Melatonin prevented the gentamicin-induced rise in plasma MDA (1.03+/-0.27 nmol) and kidney tissue MDA (2.57+/-0.87 nmol/mg protein). An increased excretion of urinary N-acetyl-beta-D-glucosaminidase, glucose, and protein by gentamicin was also prevented by melatonin. Kidneys from gentamicin-treated rats showed tubular epithelial loss with intense granular degeneration involving more than 50% of renal cortex, while there were findings comparable to controls in melatonin plus gentamicin treated rats. The present study indicates that melatonin significantly protects against gentamicin-induced renal toxicity in Wistar rats.     

Modification of catecholamine and prostaglandin tissue levels in E. coli endotoxin-treated rats

Epinephrine and norepinephrine adrenal levels were depleted in Escherichia coli endotoxin-treated (10 mg/kg) male Wistar rats from 504 +/- 203 to 119 +/- 77 ng/mg in control and from 200 +/- 97 to 123 +/- 66 ng/mg wet wt, respectively. However dopamine increased from 4.2 +/- 1.9 to 14.9 +/- 3.4 ng/mg. After endotoxin administration, norepinephrine content in peripheral organs, heart 1.27 +/- 0.19 ng/mg, spleen 1.52 +/- 0.59 ng/mg, liver 0.15 +/- 0.05 ng/mg, and kidney 0.24 +/- 0.09 ng/mg wet wt. decreased by 34, 36, 47, and 18%, respectively. Indomethacin treatment kept the catecholamine levels constant in endotoxic rats, but naloxone had no effect. PGF2 alpha tissue levels (12.0 +/- 10.1 pg/mg protein in spleen and 1.85 +/- 0.6 pg/mg protein in liver) were increased twofold by endotoxin treatment: PGE2 content in spleen and liver 0.5 +/- 0.2 pg/mg protein and 2.3 +/- 1.9 pg/mg protein, respectively, increased by only 27 and 26%. In the kidneys of endotoxin-treated animals, PGF2 alpha and PGE2 levels were lower than in control. Indomethacin treatment decreased PGF2 alpha and PGE2 and increased the norepinephrine content in the same organs. It is suggested that prostaglandins play a participative role in the control of norepinephrine tissue levels in endotoxemia.     

Effects of glucocorticoids on lung glutamine and alanine metabolism

The role of the glucocorticoid hormones as possible mediators of the accelerated lung glutamine and alanine release that occurs during critical illness was investigated. Studies were done in adult rats receiving dexamethasone (0.6 mg intramuscularly/100 gm body weight/day for 2 consecutive days; n = 24) or saline solution (controls; n = 20). Measurements were made in the postabsorptive state and amino acid flux was calculated by multiplying pulmonary blood flow by the right ventricular-arterial concentration difference for glutamine and alanine. Lung glutamine release was 703 +/- 184 nmol/100 gm body weight/min in control rats. This release rate doubled in the dexamethasone-treated rats (1476 +/- 256; p less than 0.05). The activity of the glutamine synthetase enzyme increased by 33% in the dexamethasone-treated animals and there was a 50% decrease in lung glutamine content (p less than 0.01). Likewise, dexamethasone accelerated the release of alanine by the lungs twofold (559 +/- 173 nmol/100 gm body weight/min in controls vs 1113 +/- 184 nmol/100 gm body weight/min in dexamethasone-treated rats; p less than 0.05). The increased release of both amino acids was caused by a significant increase in the concentration difference across the lungs and not a change in pulmonary blood flow. Glucocorticoids appear to be key mediators of the accelerated lung amino acid release that characterizes catabolic diseases.     

Intra-Ductal Glutamine Administration Reduces Oxidative Injury During Human Pancreatic Islet Isolation

Oxidative stress during islet isolation induces a cascade of events injuring islets and hampering islet engraftment. This study evaluated islet isolation and transplantation outcomes after intra-ductal glutamine administration. Human pancreata deemed unsuitable for pancreas or islet transplantation were treated with either a 5 mM solution of l-glutamine (n = 6) or collagenase enzyme alone (n = 6) through the main pancreatic duct. Islet yield, viability, in vitro function; markers of oxidative stress [malondialdehyde (MDA) and Glutathione (GSH)] and apoptosis were assessed. Islet yields were significantly increased in the glutamine group compared to controls (318, 559 +/- 25, 800 vs. 165, 582 +/- 39, 944 mean +/- SEM, p < 0.01). The amount of apoptotic cells per islet was smaller in the glutamine group than the control. The percentage of nude mice rendered normoglycemic with glutamine-treated islets was higher than the controls (83% n = 10/12 vs. 26% n = 6/23; p < 0.01), and the time to reach normoglycemia was decreased in the glutamine group (1.83 +/- 0.4 vs. 7.3 +/- 3 days; p < 0.01). Glutamine administration increased GSH levels (7.6 +/- 1.7 nmol/mg protein vs. 4.03 +/- 0.5 in control, p < 0.05) and reduced lipid-peroxidation (MDA 2.45 +/- 0.7 nmol/mg of protein vs. 6.54 +/- 1.7 in control; p < 0.05). We conclude that intra-ductal administration of glutamine reduces oxidative injury and apoptosis and improves islet yield and islet graft function after transplantation.     

Effect of cyclosporin A on renal function and kidney growth in the uninephrectomized rat

This study was designed to characterize the effect of cyclosporin A (CsA) on renal function and compensatory kidney growth in a rat model of uninephrectomy (Ux). The infusion of CsA (12.5 mg/k body wt) after acute Ux resulted in a fall in glomerular filtration rate (GFR) and renal plasma flow (RPF) and a marked increase in renal vascular resistance (RVR). Three weeks following Ux, GFR was also reduced in CsA treated animals as compared to pair-fed controls (0.39 +/- 0.03 vs. 0.67 +/- 0.06 ml/min/100 g, P less than 0.001), but RPF was not (1.97 +/- 0.14 vs 2.19 +/- 0.34 ml/min/100 g). The reduction in GFR seen in rats treated with CsA was fully reversible two weeks after discontinuation of the drug. Three weeks after Ux, kidney weight in CsA-treated animals increased to the level of pair-fed controls (1.50 +/- 0.05 vs. 1.57 +/- 0.06 g) but renal cortical RNA (39.4 +/- 4.3 vs. 49.3 +/- 1.3 micrograms/ml, P less than 0.05), DNA (26.4 +/- 1.7 vs. 34.7 +/- 2.1 micrograms/ml, P less than 0.01), and protein content (6.4 +/- 0.3 vs. 7.8 +/- 0.2 mg/dl, P less than 0.001) were all markedly reduced. Unilateral renal denervation in CsA-treated rats resulted in an increase in GFR and RPF as compared to that of pair-fed sham-denervated animals also treated with CsA (0.57 +/- 0.06 vs. 0.39 +/- 0.03 ml/min/100 g, P less than 0.025 and 2.14 +/- 0.14 vs. 1.63 +/- 0.20 ml/min/100 g, P less than 0.025, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Accelerated lung amino acid release in hyperdynamic septic surgical patients

Amino acid flux across the lungs was studied in humans to gain further insight into the altered nitrogen metabolism that characterizes catabolic disease states. Lung flux of glutamine, glutamate, and alanine was determined in three groups of surgical patients with indwelling pulmonary artery catheters: (1) preoperative controls (n = 14), (2) postoperative elective general surgical patients (n = 10, and (3) hyperdynamic septic surgical patients (n = 17). In controls the lung was an organ of amino acid balance. These exchange rates did not change in general surgical patients. In the septic group, glutamine release by the lung increased markedly from a control value of 0.80 +/- 0.99 mumol/kg per minute to 6.80 +/- 1.32 mumol/kg per minute. This accelerated release rate was secondary to both an increase in total pulmonary blood flow and an increase in the pulmonary artery-systemic arterial concentration difference. The lung also became an organ of significant alanine release in septic patients. The lung plays an active metabolic role in the processing of amino acids and may be a key regulator in interorgan nitrogen flux after major injury and infection.     

The effect of dexamethasone and endotoxin administration on biliary IgA and bacterial adherence.

Adherence of bacteria to the intestinal epithelial cell may be the crucial initiating event for invasion and translocation and is normally prevented by both immune (IgA) and nonimmune (mucus, peristalsis, desquamation) mucosal defense mechanisms. The purpose of the present study was to examine the effect of endotoxin administration on mucosal immunity and to define the role of glucocorticoids, commonly released during endotoxicosis, in this process. Thirty female Fisher rats were randomly assigned to three groups of 10 animals each. Group I (CONT), was fed rat chow and H2O ad lib., Group II (DEX) was administered 0.8 mg/kg subcutaneously of dexamethasone, and Group III (ETX) was given 1 mg/kg of endotoxin. Twenty-four hours later animals were sacrificed and mesenteric lymph nodes and vigorously washed stool-free ceca were collected and cultured. Bile was collected and assayed for IgA from 5 animals in each group. A significant decrease (P < 0.05) in secretory IgA was noted in animals treated with either dexamethasone or endotoxin (CONT = 332 +/- 42, DEX = 78 +/- 24, ETX = 68 +/- 16 micrograms/mg protein +/- SEM). No difference in S-IgA between animals in the dexamethasone-treated group and the endotoxin-treated group was noted (P = NS). A statistically significant increase (P < 0.001) in bacteria adherent to the cecal wall in both the dexamethasone-treated rats and the endotoxin-treated rats over that in = 7.5 +/- 0.8, CONT = 6.4 +/- 0.6 cfu/g(log10) +/- SD). Our results suggest that endotoxin or glucocorticoid administration results in significant bacterial adherence to the cecal mucosa and a decrease in IgA.(ABSTRACT TRUNCATED AT 250 WORDS)