Effects of hypertonic buffer composition on lymph node uptake and bioavailability of rituximab, after subcutaneous administration.

The subcutaneous administration of biologics is highly desirable; however, incomplete bioavailability after s.c. administration remains a major challenge. In this work we investigated the effects of excipient dependent hyperosmolarity on lymphatic uptake and plasma exposure of rituximab as a model protein. Using Swiss Webster (SW) mice as the animal model, we compared the effects of NaCl, mannitol and O-phospho-L-serine (OPLS) on the plasma concentration of rituximab over 5 days after s.c. administration. An increase was observed in plasma concentrations in animals administered rituximab in hypertonic buffer solutions, compared with isotonic buffer. Bioavailability, as estimated by our pharmacokinetic model, increased from 29% in isotonic buffer to 54% in hypertonic buffer containing NaCl, to almost complete bioavailability in hypertonic buffers containing high dose OPLS or mannitol. This improvement in plasma exposure is due to the improved lymphatic trafficking as evident from the increase in the fraction of dose trafficked through the lymph nodes in the presence of hypertonic buffers. The fraction of the dose trafficked through the lymphatics, as estimated by the model, increased from 0.05% in isotonic buffer to 13% in hypertonic buffer containing NaCl to about 30% for hypertonic buffers containing high dose OPLS and mannitol. The data suggest that hypertonic solutions may be a viable option for improving s.c. bioavailability.

Bolus injection of hypertonic solutions for cerebral edema in rats: challenge of homeostasis of healthy brain.

Hypertonic solutions are mainstay of osmotherapy to cerebral edema. How hypertonic solutions affect healthy brain homeostasis, however, is not fully understood. Using rat model of cerebral edema induced by local cryoinjury, we found with immunohistochemistry that less microglial activation in healthy hemishere 24 h after hypertonic saline (HS, 3% NaCl) administration, compared to mannitol (20%, the same osmotic concentration of 3% NaCl) while dehydrating the brain tissue. To see whether blood-brain barrier (BBB) or aquaporin-4 (AQP4) contribute to this difference, HS or mannitol was intra-arterially injected to normal rats, and BBB opening, ultrastructure and AQP4 immunoreactivity were examined. Evans blue extravasation indicated that BBB was opened much lighter in HS group than mannitol group at the same time points. Electron microscopy also showed edema around the capillaries slightly lighter in HS than mannitol group 24 h after injection. Meanwhile, HS injection led to AQP4 down regulation in expression similarly as mannitol, compared with NS group. These data suggested that bolus injection of hypertonic agents may lead to microglia activation in healthy brain in different extent, due to BBB compromise, instead of water movement or AQP4 expression. Hence in clinical application, BBB of healthy brain should be considered in perspective to maintain the brain homeostasis.

Glutamate release through connexin 43 by cultured astrocytes in a stimulated hypertonicity model.

We investigated the role of connexin 43 (Cx43) hemichannels in the release of glutamate by astrocytes after hypertonic stimulus. Mechanical, osmotic and oxidative stress, and changes in the extracellular or intracellular Ca(2+) levels induce connexin hemichannels located in the plasma membrane to open and release small ions and molecules with signaling potential such as glutamate, ATP, etc. In our past studies, we primarily found that acute hypertonic stimulus induced the release of glutamate. Since glutamate release was involved with several routes, we studied its release routes by astrocytes incubated in a hypertonic media for various periods. The glutamate release was increased after hypertonic stimulus. Glutamate release in hypertonic stimulus was inhibited by gap junction or Cx43 hemichannel blockers, but not by antagonists of purinergic receptor (P2XnR), glutamate transport inhibitors, intracellular Ca(2+) blockers, and pannexin 1(Panx1) hemichannel. The results suggest that glutamate release by the Cx43 hemichannels is likely to feature in the response of cultured astrocytes to hypertonic stimulus.

TRPV1 activation is required for hypertonicity-stimulated inflammatory cytokine release in human corneal epithelial cells.

PURPOSE: To determine whether hypertonic stress promotes increases in inflammatory cytokine release through transient receptor potential vanilloid channel type 1 (TRPV1) signaling pathway activation in human corneal epithelial cells (HCECs). METHODS: Hyperosmotic medium was prepared by supplementing isotonic Ringers solution with sucrose. Ca2+ signaling was measured in fura2-AM-loaded HCECs using a single-cell fluorescence imaging system. Western blot analysis evaluated the phosphorylation status of EGFR, ERK, p38 MAPK, and nuclear factor (NF)-κB. ELISA assessed the effect of TRPV1 activation on the release of IL-6 and IL-8. RESULTS: A 450 mOsm hypertonic stress elicited 2-fold Ca2+ transients that were suppressed by the TRPV1-selective antagonists capsazepine and JYL 1421. Such transients were enhanced by PGE2. Hypertonicity-induced EGF receptor (EGFR) transactivation was suppressed by preincubating HCECs with capsazepine, matrix metalloproteinase 1 (MMP1) inhibitor TIMP-1, broad-spectrum MMP inhibitor GM 6001, heparin-bound (HB)-EGF inhibitor CRM 197, or EGFR inhibitor AG 1478. ERK and p38 MAPK and NF-κB activation after EGFR transactivation occurred in tonicity and in a time-dependent manner. Hypertonicity-induced increases in IL-6 and IL-8 releases were suppressed by exposure to capsazepine, AG 1478, ERK inhibitor PD 98059, p38 inhibitor SB 203580, or NF-κB inhibitor PDTC. CONCLUSIONS: Hypertonic stress-elicited TRPV1 channel stimulation mediates increases in a proinflammatory cytokine IL-6 and a chemoattractant IL-8 by eliciting EGFR transactivation, MAPK, and NF-κB activation. Selective drug modulation of either TRPV1 activity or its signaling mediators may yield a novel approach to suppressing inflammatory responses occurring in dry eye syndrome.

The effect of pinealectomy on plasma vasopressin response to isotonic, hypertonic and hypovolemic treatments in rats supplemented with L-thyroxine.

The present study was conducted to determine the effects of basal, isotonic as well as hypertonic and hypovolemic treatments on fluid-electrolyte balance and plasma AVP levels in rats supplemented with L-thyroxine and pinealectomized L-thyroxine. The animals were initially separated into 4 groups: control (n = 24), L-thyroxine treated (n = 24); L-thyroxine + sham-pinealectomy (n = 24) treated and 4-L-thyroxine + pinealectomy (n = 24) treated. L-thyroxine was given for 4 weeks. At the end of the 4-week experimental period, the sub-groups were formed before decapitation, which were classified as unstimulated (n = 6), isotonic (n = 6), hypertonic (n = 6) and hypovolemic (n = 6) stimulation. Plasma AVP, total triiodothyronine (TT3) and total thyroxine (TT4) levels were examined in plasma by RIA. Hematocrit and osmolality levels were also determined. It was found that the TT3 and TT4 levels showed significant increases in L-thyroxine treated groups (P <0.001). Also, plasma AVP levels increased in the group subjected to L-thyroxine treatment. However, this increase was depicted to be significantly more prominent in L-thyroxine + pinealectomy treated group (P <0.001). The results of the present study indicate that L-thyroxine treatment increases the basal and stimulated AVP release, which became more significant in the pinealectomy plus L-thyroxine treatment group. Moreover, the results indicate that AVP response to hypertonic and hypovolemic stimulations does not undergo any change due to supplementation by L-thyroxine treatment and/or pinealectomy plus L-thyroxine.

Regulatory and necrotic volume increase in boar spermatozoa.

Spermatozoa of many species initially respond to hypotonicity as perfect osmometers. Thereafter they undergo a regulatory process resulting in a decrease in cell volume, similar to that reported for somatic cells. Regulatory volume increase (RVI), a complementary process which is assumed to occur following initial shrinkage of sperm volume after exposure to a hypertonic medium, has not yet been described in detail for spermatozoa. In this study, we investigated whether spermatozoa are able to regulate their volume after hypertonic stress and whether this ability is maintained in preserved sperm. Cell volume changes were recorded using electronic cell sizing. Sperm response to the ion channels blockers quinidine, tamoxifen, and dydeoxyforskolin, and to protein kinase/phosphatase inhibitors lavendustin, staurosporine, and vanadate was studied to investigate possible mechanisms of RVI. Annexin V staining was used in combination with propidium iodide to determine whether hypertonic stress may induce apoptosis. Overall protein tyrosine phosphorylation under hypertonic conditions was measured via flow cytometry using antiphosphotyrosine antibody. Spermatozoa exposed to hypertonic stress initially responded with an abundant subpopulation according to the perfect osmometer model and recovered their volume from this shrinkage after 20 min. RVI was inhibited by quinidine and tamoxifen, which indicates the involvement of the important cellular ions sodium and chloride in this process. Volume regulatory ability was essentially maintained during storage of liquid semen. However, the response of the sperm population was heterogeneous. A second population raised, containing spermatozoa with larger volumes, which demonstrated irregularities in the volume response with respect to osmotic challenge, ion channel blockers, and storage. Under hypertonic conditions, both protein kinase inhibitors (PKI) led to increased isotonic volumes and to elevated initial relative volumes and subsequent volume decrease. RVI was inhibited by the vanadate. Hypertonic stress did not result in an increase in early apoptotic cells, but produced a shift toward late necrotic cells. Substitution of sodium and chloride by choline and sulfate resulted in decreased isotonic volume of sperm treated with lavendustin. Tyrosine phosphorylation levels were reduced after 20 min under hypertonic conditions. It was concluded that RVI is regulated via a protein tyrosine kinase-dependent pathway, and that dephosphorylation occurs when volume regulation is required. The necrotic volume increase (NVI) is associated with the accumulation of sodium and chloride following uncontrolled opening of the channels. The ability to regulate volume after exposure to hypertonic conditions is important for sperm functionality and can have practical applications in spermatological diagnostics and cryopreservation.

The new approaches to preservation of graft cell integrity in preservation for transplantation.

Restoration of cell plasma membrane integrity after injury is essential for the survival of animal cells. In case of graft preservation or during chemotherapy in cancer, cell membrane integrity and the process of its repair are disrupted. Cytoprotective substances are important in such cases, as well as in other diseases, for example in myocardial infarction, acute insults and in chronic neurodegenerative diseases. Hyperosmolarity is a condition in which cell membrane stability may be damaged in vivo but preserved in the in vitro conditions. Hypertonicity causes water leaving from cells by osmosis, decreasing cell volume and increasing of intracellular ionic strength. High intracellular ionic strength perturbs cellular function by decreasing the rates of biochemical reaction. We review the new experimentally studied cytoprotective substances and their application in cell membrane protection. Moreover, we present our data on the effects of hyperosmolarity and its protective effect on cell internal structure.

The synthesis of SNAT2 transporters is required for the hypertonic stimulation of system A transport activity.

In cultured human fibroblasts incubated under hypertonic conditions, the stimulation of system A for neutral amino acid transport, associated to the increased expression of the mRNA for SNAT2 transporter, leads to an expanded intracellular amino acid pool and to the recovery of cell volume. A protein of nearly 60 kDa, recognized by an antiserum against SNAT2, is increased both in the pool of biotinylated membrane proteins and in the total cell lysate of hypertonically stressed cells. The increased level of SNAT2 transporters in hypertonically stressed cells is confirmed by immunocytochemistry. DRB, an inhibitor of transcription, substantially inhibits the increase of SNAT2 proteins on the plasma membrane, completely suppresses the stimulation of system A transport activity, and markedly delays the cell volume recovery observed during the hypertonic treatment. On the contrary, if the transport activity of system A is adaptively increased by amino acid starvation in the presence of DRB, the increase of SNAT2 transporters on the plasma membrane is still clearly detectable and the transport change only partially inhibited. It is concluded that the synthesis of new SNAT2 transporters is essential for the hypertonic stimulation of transport system A, but accounts only in part for the adaptive increase of the system.

Nicotine enhancement of dopamine release by a calcium-dependent increase in the size of the readily releasable pool of synaptic vesicles.

A major factor underlying compulsive tobacco use is nicotine-induced modulation of dopamine release in the mesolimbic reward pathway (Wise and Rompre, 1989). An established biochemical mechanism for nicotine-enhanced dopamine release is by activating presynaptic nicotinic acetylcholine receptors (nAChRs) (Wonnacott, 1997). Prolonged application of 10(-7) to 10(-5) m nicotine to striatal synaptosomes promoted a sustained efflux of [3H]dopamine. This nicotine effect was mediated by non-alpha7 nAChRs, because it was blocked by 5 mum mecamylamine but was resistant to 100 nm alpha-bungarotoxin (alphaBgTx). Dopamine release was diminished by omitting Na+ or by applying peptide calcium channel blockers, indicating that nAChRs trigger release by depolarizing the nerve terminals. However, because alpha7 receptors rapidly desensitize in the continuous presence of agonists, a repetitive stimulation protocol was used to evaluate the possible significance of desensitization. This protocol produced a transient increase in [3H]dopamine released by depolarization and a significant increase in the response to hypertonic solutions that measure the size of the readily releasable pool (RRP) of synaptic vesicles. The nicotine-induced increase in the size of the readily releasable pool was blocked by alphaBgTx and by the calmodulin antagonist calmidazolium, suggesting that Ca2+ entry through alpha7 nAChRs specifically enhances synaptic vesicle mobilization at dopamine terminals. Thus, nicotine enhances dopamine release by two complementary actions mediated by discrete nAChR subtypes and suggest that the alpha7 nAChR-mediated pathway is tightly and specifically coupled to refilling of the RRP of vesicles in dopamine terminals.

The organum vasculosum laminae terminalis in immune-to-brain febrigenic signaling: a reappraisal of lesion experiments.

The organum vasculosum laminae terminalis (OVLT) has been proposed to serve as the interface for blood-to-brain febrigenic signaling, because ablation of this structure affects the febrile response. However, lesioning the OVLT causes many "side effects" not fully accounted for in the fever literature. By placing OVLT-lesioned rats on intensive rehydration therapy, we attempted to prevent these side effects and to evaluate the febrile response in their absence. After the OVLT of Sprague-Dawley rats was lesioned electrolytically, the rats were given access to 5% sucrose for 1 wk to stimulate drinking. Sucrose consumption and body mass were monitored. The animals were examined twice a day for signs of dehydration and treated with isotonic saline (50 ml/kg sc) when indicated. This protocol eliminated mortality but not several acute and chronic side effects stemming from the lesion. The acute effects included adipsia and gross (14% of body weight) emaciation; chronic effects included hypernatremia, hyperosmolality, a suppressed drinking response to hypertonic saline, and previously unrecognized marked (by approximately 2 degrees C) and long-lasting (>3 wk) hyperthermia. Because the hyperthermia was not accompanied by tail skin vasoconstriction, it likely reflected increased thermogenesis. After the rats recovered from the acute (but not chronic) side effects, their febrile response to IL-1beta (500 ng/kg iv) was tested. The sham-operated rats developed typical monophasic fevers ( approximately 0.5 degrees C), the lesioned rats did not. However, the absence of the febrile response in the OVLT-lesioned rats likely resulted from the untreatable side effects. For example, hyperthermia at the time of pyrogen injection was high enough (39-40 degrees C) to solely prevent fever from developing. Hence, the changed febrile responsiveness of OVLT-lesioned animals is given an alternative interpretation, unrelated to febrigenic signaling to the brain.

Release of ATP induced by hypertonic solutions in Xenopus oocytes.

ATP mediates intercellular communication. Mechanical stress and changes in cell volume induce ATP release from various cell types, both secretory and non-secretory. In the present study, we stressed Xenopus oocytes with a hypertonic solution enriched in mannitol (300 mM). We measured simultaneously ATP release and ionic currents from a single oocyte. A decrease in cell volume, the activation of an inward current and ATP release were coincident. We found two components of ATP release: the first was associated with granule or vesicle exocytosis, because it was inhibited by tetanus neurotoxin, and the second was related to the inward current. A single exponential described the correlation between ATP release and the hypertonic-activated current. Gadolinium ions, which block mechanically activated ionic channels, inhibited the ATP release and the inward current but did not affect the decrease in volume. Oocytes expressing CFTR (cystic fibrosis transmembrane regulator) released ATP under hypertonic shock, but ATP release was significantly inhibited in the first component: that related to granule exocytosis. Since the ATP measured is the balance between ATP release and ATP degradation by ecto-enzymes, we measured the nucleoside triphosphate diphosphohydrolase (NTPDase) activity of the oocyte surface during osmotic stress, as the calcium-dependent hydrolysis of ATP, which was inhibited by more than 50 % in hypertonic conditions. The best-characterized membrane protein showing NTPDase activity is CD39. Oocytes injected with an antisense oligonucleotide complementary to CD39 mRNA released less ATP and showed a lower amplitude in the inward current than those oocytes injected with water.

Hyperosmolarity reduces facilitation by a Ca(2+)-independent mechanism at the lobster neuromuscular junction: possible depletion of the releasable pool.

1. At the crustacean neuromuscular junction, action potential-evoked neurosecretion increases in proportion to stimulation frequency, a process termed frequency facilitation. In the present study we examined how frequency facilitation is affected by osmotic pressure. 2. Hypertonic solution (HS) was applied by local superfusion of the synaptic area. Quantal release was monitored by focal extracellular recordings of postsynaptic potentials. Several stimulation frequencies (f) in the range from 1 to 10 Hz were employed, and quantal content (m) together with the number of releasable units (n) and release probability (p) was evaluated for each frequency. 3. Osmotic pressure enhanced quantal release at the lowest f tested (1 Hz) but suppressed neurosecretion at higher f (7-10 Hz). Thus, hyperosmolarity enhanced action potential-evoked release but suppressed frequency facilitation. 4. Chelation of intracellular calcium by BAPTA showed that the effect of HS was calcium independent. 5. Binomial analysis of quantal content revealed that HS suppressed the increase in the number of releasable units, which was very pronounced during facilitation under control conditions. Since HS also stimulated asynchronous quantal release, the observed effect of HS on facilitation can be explained by the depletion of the releasable pool of quanta caused by the asynchronous neurosecretion. 6. To test this hypothesis we increased the available pool of vesicles using serotonin and demonstrated that the suppressing effect of HS on facilitation was reversed. 7. The observed effects of HS on facilitated neurosecretion could be described quantitatively using our model for mobilization of vesicles into the releasable pool enhanced by action potentials.

Appearance of central dipsogenic mechanisms induced by dehydration in near-term rat fetus.

Cellular dehydration of central osmoreceptors evokes an integration of behavioral (i.e. drinking) and endocrinologic (i.e. arginine vasopressin secretion) responses to maintain body fluid balance. These osmoregulatory mechanisms have been intensely investigated in adult models. However, there has been limited research of the fetal development of neural mechanisms regulating responses to dehydration. Although behavioral and neuroendocrine responses to dehydration have been demonstrated in utero in precocial species (e.g. ovine), there has been no study to date demonstrating that these responses develop before the neonatal period of altricial species (e.g. rat). This study is the first to use the near-term rat fetus to investigate the effects of maternal subcutaneous hypertonic (2 M NaCl) or isotonic (0.15 M NaCl) saline injection on fetal plasma osmolality and brain FOS-immunoreactivity (FOS-ir). Maternal subcutaneous hypertonic saline significantly increased maternal and fetal plasma osmolality to similar levels (328+/-6 and 326+/-6 mosM/kg, respectively). In response to plasma hypertonicity, maternal and fetal brain FOS-ir increased significantly in the regions including the lamina terminalis, and the supraoptic and paraventricular nuclei (SON and PVN) of the hypothalamus. Together, these data indicate that central mechanisms for dipsogenic and arginine vasopressin secretory responses to hypertonicity are present and responsive in the fetal rat brain at near-term gestation. However, differences between fetal and maternal FOS-ir mapping suggest that fetal osmoreceptor development is not yet completed near term.

Vasopressin, renin, and adrenocorticotropic hormone levels during the resuscitation of hemorrhagic shock in dogs.

Hemorrhage is a potent stimulus for the release of vasopressin (VP), renin, and adrenocorticotropic hormone (ACTH). The goal of this study was to analyze changes in plasma VP, renin, and ACTH levels during hemorrhagic shock and resuscitation with two different solutions: hypertonic acetate dextran (HAD) and lactated Ringer's (LR) solution. Eight randomized dogs were shocked by removing 37 +/- 9 mL/kg of blood while maintaining the mean arterial pressure (MAP) at 45 +/- 5 mmHg for 1 h. Test solutions were randomized and infused as needed with the hemorrhaged blood to restore the MAP and cardiac index to baseline. Blood samples for hormone analyses were taken in baseline, shock, and resuscitation periods. For each experiment, all hormone levels increased in the postshock period and then returned to baseline values after resuscitation with both solutions. VP and renin levels rapidly returned to baseline values after resuscitation in the LR dogs compared with the HAD dogs (p < 0.05). By contrast, there was no significant difference in ACTH levels between the two solutions. High-volume infusion with LR achieves more rapid restoration than small-volume infusion with HAD for VP and renin levels.

Superior protection in orthotopic rat lung transplantation with cyclic adenosine monophosphate and nitroglycerin-containing preservation solution.

BACKGROUND: Primary lung graft failure is common, and current lung preservation strategies are suboptimal. Because the decline in lung levels of cyclic adenosine monophosphate and cyclic guanosine monophosphate during preservation could enhance adhesiveness of endothelial cells for leukocytes as well as increase vascular permeability and vasoconstriction, we hypothesized that buttressing these levels by means of a preservation solution would significantly improve lung preservation. METHODS: An orthotopic rat left lung transplantation model was used. Lungs were harvested from male Lewis rats and preserved for 6 hours at 4 degrees C with (1) Euro-Collins solution (n = 8); (2) University of Wisconsin solution (n = 8); (3) low-potassium dextran glucose solution (n = 8); (4) Columbia University solution (n = 8), which contains a cyclic adenosine monophosphate analog (dibutyryl cyclic adenosine monophosphate) and a nitric oxide donor (nitroglycerin) to buttress cyclic guanosine monophosphate levels; or (5) Columbia University solution without cyclic adenosine monophosphate or nitroglycerin (n = 8). PaO2, pulmonary vascular resistance, and recipient survival were evaluated 30 minutes after left lung transplantation and removal of the nontransplanted right lung from the pulmonary circulation. RESULTS: Among all groups studied, grafts stored with Columbia University solution demonstrated the highest Pa O2 (355 +/- 25 mm Hg for Columbia University solution versus 95 +/- 22 mm Hg for Euro-Collins solution, P <.01, 172 +/- 55 mm Hg for University of Wisconsin solution, P <.05, 76 +/- 15 mm Hg for low-potassium dextran glucose solution, P <.01, and 82 +/- 25 mm Hg for Columbia University solution without cyclic adenosine monophosphate or nitroglycerin, P <.01) and the lowest pulmonary vascular resistances (1 +/- 0.2 mm Hg * mL-1 * min-1 for Columbia University solution versus 12 +/- 4 mm Hg * mL-1 * min-1 for Euro-Collins solution, P <.01, 9 +/- 2 mm Hg * mL-1 * min-1 for University of Wisconsin solution, 14 +/- 6 mm Hg * mL-1 * min-1 for low-potassium dextran glucose solution, P <.01, and 8 +/- 2 mm Hg * mL-1 * min-1 for Columbia University solution without cyclic adenosine monophosphate and nitroglycerin). These functional and hemodynamic improvements provided by Columbia University solution were accompanied by decreased graft leukostasis and decreased recipient tumor necrosis factor alpha and interleukin 1alpha levels compared with the other groups. In toto, these improvements translated into superior survival among recipients of Columbia University solution-preserved grafts (100% for Columbia University solution, 37% for Euro-Collins solution, P <.01, 50% for University of Wisconsin solution, P <.05, 50% for low-potassium dextran glucose solution, P <.05, and 13% for Columbia University solution without cyclic adenosine monophosphate and nitroglycerin, P <.01). CONCLUSION: Nitroglycerin and cyclic adenosine monophosphate confer beneficial vascular effects that make Columbia University solution a superior lung preservation solution in a stringent rat lung transplantation model.

High water intake ameliorates tubulointerstitial injury in rats with subtotal nephrectomy: possible role of TGF-beta.

BACKGROUND: It has been shown that tubulointerstitial injury correlates well with a decline of renal function. In this study, we investigated the effect of high water intake (HWI) on functional and structural parameters in rats with subtotal nephrectomy. METHODS: Two weeks after the ablative procedure, rats were divided into two groups. One group received the treatment with HWI (3% sucrose added to drinking water) for eight weeks. Functional parameters were compared with sham-operated control (CONT) or nephrectomized rats without treatment (NX). Remnant kidneys were then assessed histologically for evidence of interstitial fibrosis and glomerulosclerosis. RESULTS: Creatinine clearance was significantly improved in HWI rats compared with NX rats. Simultaneously, urinary protein was also significantly reduced in HWI rats. HWI predominantly ameliorated interstitial lesions and, to a lesser extent, glomerular lesions. Northern blot analysis demonstrated that transforming growth factor-beta (TGF-beta) mRNA expression was significantly suppressed in HWI rats. In situ hybridization revealed that HWI suppressed TGF-beta mRNA expression mainly in the outer medulla. Fibronectin mRNA was also reduced by the HWI treatment. The changes in TGF-beta and fibronectin mRNA were in parallel with Na+/myo-inositol cotransporter (SMIT) mRNA, which is regulated by extracellular osmolarity. Immunohistochemistry demonstrated that protein expression of TGF-beta and fibronectin coincided with the mRNA expression. CONCLUSION: These results suggest that HWI reduces TGF-beta mRNA expression in medullary interstitium and ameliorates tubulointerstitial injury in rats with reduced renal mass.

Comparative study of solutions for pulmonary preservation using an isolated rabbit lung model.

At the University of Minnesota, University of Wisconsin (UW), modified Euro-Collins (MEC), and Marshall (M) solutions were compared as agents for pulmonary preservation in an isolated rabbit lung model. Normal saline (NS) was used as a control. The heart-lung blocks of donor rabbits were flushed with, and then preserved in, one of the solutions at 4 degrees C. Five rabbits were studied in each group. After 8 h of cold ischemia, the left lung was ventilated and reperfused with fresh venous blood from donor rabbits for 30 min. Pulmonary function was assessed by serial measurements of oxygen (O2) and carbon dioxide (CO2) tensions in blood obtained from the left atrial appendage. The ratios of wet/dry (W/D) weight of the lungs were calculated to assess the extent of pulmonary edema. After 8 h of preservation followed by 30 min of reperfusion, O2 tension was significantly higher with UW (178.36 + 1.72 mmHg). The calculated P values were UW vs NS, < 0.0001; UW vs MEC, 0.154; and UW vs M, 0.0001. CO2 tension with UW was also lower than the other solutions: UW, 35.8 +/- 0.698 mmHg; NS, 48.5 +/- 0.745 mmHg; MEC, 40.69 +/- 0.749 mmHg; and M, 44.68 +/- 0.697 mmHg. The calculated P value was UW vs NS, 0.0001; UW vs MEC, 0.0003; and UW vs M, 0.0001 using repeated-measures analysis of covariance. The W/D ratio was lower with UW as well; UW, 6.82 +/- 0.19; NS, 8.01 +/- 0.23; MEC, 7.28 +/- 0.10; and M, 7.34 +/- 0.17. The P value was < 0.001 using post-hoc tests. In this model, UW solution preserved the lungs better than the other three solutions tested and therefore warrants further clinical application.

[Intestinal lavage solution for orthograde intestinal irrigation]. / Darmspüllösung zur orthograden Darmspülung.

Gut lavage by ingestion of large volumes of electrolyte solutions has been shown to be an effective method of cleansing the colon before colonoscopy, barium enema or surgery. Absorption of water and electrolytes, which might be hazardous to patients who are unable to readily excrete an additional sodium and/or water load, is prevented by addition of non-absorbable substances to the solutions, but systematic studies are lacking. We have evaluated the influence of three solutions for gut lavage with different electrolyte composition (sodium concentration 67 mmol/l and 125 mmol/l) and addition of different non-absorbable substances (mannitol and polyethylene glycol [PEG]) on water and electrolyte homeostasis and subjective tolerance, both in healthy volunteers and in patients before endoscopy of the colon. In a randomized, blind study 6 liters of the three solutions were administered via a nasogastric tube to 6 healthy volunteers during 4 hours (i.e. 1.5 l/h). Body weight, serum concentrations of sodium, potassium and of phosphate were measured before infusion of the solution and after the last rhythmic rectal effluent. No significant changes were observed in any of the studied parameters and the incidence of side effects (nausea, abdominal cramps) was comparable. In an additional clinical double blind study, 26 patients before diagnostic colonoscopy were asked to drink 4 liters of the gut lavage solutions as quickly as possible in order to clean out the colon. The time for drinking was significantly shorter in patients using the mannitol and low sodium solution (204 +/- 70 minutes) than in patients drinking the solution with polyethylene glycol and a high sodium concentration (387 +/- 137 minutes). There was a tendency to a longer drinking period in patients ingesting the solution with polyethylene glycol and low sodium (306 +/- 106 minutes). Thus, the acceptance for solutions containing polyethylenglycol and high sodium concentration is reduced because of low palatibility. Again no influence on serum electrolyte concentrations or body weight could be observed in any patient, the spectrum of side effects was similar and the cleansing effect of all three solutions was adequate. In conclusion solutions for gut lavage containing a balanced electrolyte concentration and nonresorbable substances such as mannitol or polythylenglycol are equivalent. However, solutions containing mannitol and a low sodium concentration are better tolerated by the patients but the use of mannitol is limited because of the risk of releasing explosive gases during interventional endoscopy. To enhance the acceptance and palatibility of solutions for gut lavage containing polethylenglycol the addition of flavoured substances is recommended.

Hypertonicity induces injury to cultured human endothelium: attenuation by glutamine.

BACKGROUND: Although most preservation solutions as well as some cardioplegic solutions used for organ storage and transplantation are hypertonic, the effects of extracellular hypertonicity on endothelium are not well established. Aims of this study were to evaluate the response of cultured human saphenous vein endothelial cells to extracellular hypertonicity and to investigate the role of the amino acid glutamine in preventing endothelial damage in vitro. METHODS: Eight distinct strains of human saphenous vein endothelial cells were studied. Hypertonic (350 and 400 mosm/kg) media were obtained by supplementing culture medium with sucrose. Cell viability was assessed in the absence or the presence of glutamine through the determination of cell number and protein content of the cultures. Confocal microscopy of cells loaded with the fluorescent dye calcein was also performed. RESULTS: Exposure of human saphenous vein endothelial cells to hypertonic media without glutamine caused significant cell loss within 30 minutes. Cell loss progressed steadily during incubation and after 6 hours reached 50% at 350 mosm/kg and 65% at 400 mosm/kg. In the presence of 2 mmol/L glutamine, endothelial damage was completely prevented at 350 mosm/kg and significantly lessened at 400 mosm/kg compared with glutamine-free media. Confocal microscopy showed that most hypertonicity-treated cells exhibited the typical features of an apoptotic death and confirmed the osmoprotective effect of glutamine. CONCLUSIONS: These results indicate that the supplementation of hypertonic storage solutions with glutamine might exert a partial osmoprotective effect and suggest that the relationship between endothelial damage and tonicity of storage and cardioplegic solutions should be carefully investigated.

Transient expression of Na+/H+ exchanger isoform NHE-2 in LLC-PK1 cells: inhibition of endogenous NHE-3 and regulation by hypertonicity.

Na+/H+ exchanger isoforms NHE-2 and NHE-3 demonstrate distinct tissue expression patterns in renal epithelial cells. NHE-2 is predominantly expressed in the inner medulla whereas NHE-3 is highly expressed in the proximal tubule cells. The purpose of the current experiments was to study the characteristics of NHE-2 upon its own expression in cultured proximal tubule cells, LLC-PK1. Toward this end, LLC-PK1 cells were subjected to six cycles of proton suicide. The mutant cells, when grown to confluence and assayed for Na+/H+ exchanger by 22Na+ influx, showed significant reduction in NHE activity as compared to the parent cells (10.4 nmole/mg prot/4 min in parent cells vs. 1.8 in mutant cells, P < 0.001, n = 4). This remaining exchanger activity was mostly mediated via NHE-3 as shown by inhibition of the Na influx following PKC stimulation (65% with PMA vs. 100% without PMA. P < 0.05, n = 4). The mutant cells were transiently transfected with a pCMV/NHE-2 expression vector using calcium phosphate precipitation method. Northern blot analysis showed the expression of a 3.4 kb transcript only in the transfected cells. The expression peaked at 48 hr and diminished by 96 hr. The exchanger activity at 48 hr after transfection was mostly due to NHE-3 (as shown by inhibition in the presence of PMA) but was significantly lower than in sham transfected cells (1.2 nmoles/mg prot. in NHE-2-transfected and 2.1 in sham-transfected, P < 0.05, n = 4). At 60 hr after transfection, the cells exhibited PMA-stimulated Na influx (>28%) indicating functional expression of NHE-2. Increasing the osmolality of the media to 510 mOsm/l stimulated the Na+/H+ exchanger in NHE-2 transfected cells but inhibited the exchanger activity in sham transfected cells. In conclusion, NHE-2 appears as a 3.4 kb transcript in transfected LLC-PK1 cells and functional expression of NHE-2 is preceded by inhibition of endogenous NHE-3 activity. The NHE-2 is stimulated by hypertonicity, indicating a likely role for this isoform in cell volume regulation.