Cotransport System Research Articles

Guanylin activates Cl(-) secretion into the lumen of seawater eel intestine via apical Cl(-) channel under simulated in vivo conditions.

Guanylin (GN) action on seawater eel intestine was examined under simulated in vivo conditions, where isotonic luminal fluid has low NaCl and high MgSO4 (MgSO4 Ringer). In Ussing chamber, MgSO4 Ringer induced serosa-negative potential difference (PD) even after bumetanide treatment, which is due to the higher paracellular Na(+) permeability over Cl(-), as confirmed by the replacement by MgCl2 (no Cl(-) gradient) or Na2SO4 Ringer (no Na(+) gradient). Luminal GN reversed serosa-negative PD, probably by enhancing Cl(-) secretion into the lumen, as the GN effect was blocked by apical Cl(-) channel blockers [diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino) benzoic acid, glibenclamide but not cystic fibrosis transmembrane regulator (CFTR)inh-172] or replacement of luminal fluid by MgCl2 Ringer. The blockers' effect was undetectable when normal Ringer was on both sides. In the sac preparation, NaCl secretion occurred into the lumen (Na(+) > Cl(-)), and GN further enhanced Cl(-) secretion (Cl(-) > Na(+)), resulting in water secretion. These GN effects were also blocked by DPC. Quantitative analyses showed that isotonic NaCl is absorbed when luminal fluid is normal Ringer, but, when luminal fluid is MgSO4 Ringer, hypertonic NaCl, almost equivalent to seawater, is secreted into the lumen after GN. These results indicate that GN stimulates the secretion of hypertonic NaCl into the lumen of seawater eel intestine, like rectal gland of marine elasmobranchs, to get rid of excess NaCl although marine teleost intestine is thought to have only absorptive-type cells with a unique Na-K-Cl cotransport system. The secreted NaCl may activate the cotransport system and further help absorb water in the final segment of seawater eel intestine.

Dynamics of sodium and potassium in plasma, erythrocytes, and muscles of freshwater species under the effect of long-term combined stress

In the initial period of acute stress, the concentration of Na in the plasma of roach and bream decreases by 30 and 70% in several hours, thereby causing a difference in the osmotic concentration of cellular and extracellular liquids of an organism. In response to this, defensive systems are activated. They have extracted Na and K ions from the cell against their electrochemical gradient. As a result, the content of Na and K in erythrocytes and muscles of fish has decreased to a minimum. During the acclimatization of fish to new conditions, the ion concentration in plasma, erythrocytes, and muscles has stabilized at low levels, reflecting the physiological state of the organism. It is assumed that the counteraction of hyponatremia and hypoosmolality has been achieved due to the intensification of the Na+-K+-2Cl− cotransport system.

Role of AE2 for pHi regulation in biliary epithelial cells

The Cl−/HCO−3anion exchanger 2 (AE2) is known to be involved in intracellular pH (pHi) regulation and transepithelial acid-base transport. Early studies showed that AE2 gene expression is reduced in liver biopsies and blood mononuclear cells from patients with primary biliary cirrhosis (PBC), a disease characterized by chronic non-suppurative cholangitis associated with antimitochondrial antibodies (AMA) and other autoimmune phenomena. Microfluorimetric analysis of the Cl−/HCO−3 anion exchange (AE) in isolated cholangiocytes showed that the cAMP-stimulated AE activity is diminished in PBC compared to both healthy and diseased controls. More recently, it was found that miR-506 is upregulated in cholangiocytes of PBC patients and that AE2 may be a target of miR-506. Additional evidence for a pathogenic role of AE2 dysregulation in PBC was obtained with Ae2−/−a,b mice, which develop biochemical, histological, and immunologic alterations that resemble PBC (including development of serum AMA). Analysis of HCO−3 transport systems and pHi regulation in cholangiocytes from normal and Ae2−/−a,b mice confirmed that AE2 is the transporter responsible for the Cl−/HCO−3exchange in these cells. On the other hand, both Ae2+/+a,b and Ae2−/−a,b mouse cholangiocytes exhibited a Cl−-independent bicarbonate transport system, essentially a Na+-bicarbonate cotransport (NBC) system, which could contribute to pHi regulation in the absence of AE2.

Transport of l-proline by the proton-coupled amino acid transporter PAT2 in differentiated 3T3-L1 cells

Mechanism and substrate specificity of the proton-coupled amino acid transporter 2 (PAT2, SLC36A2) have been studied so far only in heterologous expression systems such as HeLa cells and Xenopus laevis oocytes. In this study, we describe the identification of the first cell line that expresses PAT2. We cultured 3T3-L1 cells for up to 2 weeks and differentiated the cells into adipocytes in supplemented media containing 2 μM rosiglitazone. During the 14 day differentiation period the uptake of the prototype PAT2 substrate L-[(3)H]proline increased ~5-fold. The macro- and microscopically apparent differentiation of 3T3-L1 cells coincided with their H(+) gradient-stimulated uptake of L-[(3)H]proline. Uptake was rapid, independent of a Na(+) gradient but stimulated by an inwardly directed H(+) gradient with maximal uptake occurring at pH 6.0. L-Proline uptake was found to be mediated by a transport system with a Michaelis constant (K(t)) of 130 ± 10 μM and a maximal transport velocity of 4.9 ± 0.2 nmol × 5 min(-1 )mg of protein(-1). Glycine, L-alanine, and L-tryptophan strongly inhibited L-proline uptake indicating that these amino acids also interact with the transport system. It is concluded that 3T3-L1 adipocytes express the H(+)-amino acid cotransport system PAT2.

Loop diuretics have anxiolytic effects in rat models of conditioned anxiety.

A number of antiepileptic medications that modulate GABAA mediated synaptic transmission are anxiolytic. The loop diuretics furosemide (Lasix) and bumetanide (Bumex) are thought to have antiepileptic properties. These drugs also modulate GABAA mediated signalling through their antagonism of cation-chloride cotransporters. Given that loop diuretics may act as antiepileptic drugs that modulate GABAergic signalling, we sought to investigate whether they also mediate anxiolytic effects. Here we report the first investigation of the anxiolytic effects of these drugs in rat models of anxiety. Furosemide and bumetanide were tested in adult rats for their anxiolytic effects using four standard anxiety models: 1) contextual fear conditioning; 2) fear-potentiated startle; 3) elevated plus maze, and 4) open-field test. Furosemide and bumetanide significantly reduced conditioned anxiety in the contextual fear-conditioning and fear-potentiated startle models. At the tested doses, neither compound had significant anxiolytic effects on unconditioned anxiety in the elevated plus maze and open-field test models. These observations suggest that loop diuretics elicit significant anxiolytic effects in rat models of conditioned anxiety. Since loop diuretics are antagonists of the NKCC1 and KCC2 cotransporters, these results implicate the cation-chloride cotransport system as possible molecular mechanism involved in anxiety, and as novel pharmacological target for the development of anxiolytics. In view of these findings, and since furosemide and bumetanide are safe and well tolerated drugs, the clinical potential of loop diuretics for treating some types of anxiety disorders deserves further investigation.

Evidence for Activation of AMP Kinase (AMPK) in Nephropathic Cystinosis

The objective of this study was to investigate the involvement of AMPK in the altered signaling that results in nephropathic cystinosis. Cystinosis is characterized by cysteine accumulation, due to mutations in CTNS, encoding for cystinosin, a lysosomal cystine transporter. Patients develop the Fanconi Syndrome, due to defective reabsorption by apical Na+/solute cotransport systems in the renal proximal tubule (RPT). In this study, cystinosin was knocked down >80% in primary rabbit RPT cells using cystinosin siRNA. This reduction in cystinosin strongly inhibited Na+ dependent Phosphate (Pi) uptake (70%). Intracellular Na+and Na,K‐ATPase were unaffected. However, ATP dropped 52%, the ADP/ATP ratio increased 300%, and apical NaPi2a transporters were reduced. Because increases in ADP result in AMPK phosphorylation and activation, the phosphorylated AMPKα subunit (pAMPKα) was examined. In primary RPT cells with a cystinosin knockdown the level of pAMPKα was elevated (8.0 +/− 4.2 fold relative to the control), and was unaffected by AICAR (5‐Aminoimidazole‐4‐Carboxamide‐1‐β‐D‐ribofuranoside), an AMPK activator (unlike control cells). Further studies are in progress to determine whether AMPK activation is responsible for the reduced growth, increased apoptotis, as well as reduced transport in primary RPT cells with a cystinosin knockdown. The funding was from the Cystinosis Research Foundation.

Bumetanide

Bumetanide

Design, synthesis and biological activity of a series of torasemide derivatives, potent blockers of the Na+ 2Cl- K+ co-transporter: in-vitro study.

Pharmacomodulation of the torasemide molecule, a loop diuretic inhibiting Na+ 2Cl- K+ co-transport in the thick ascending limb of the loop of Henlé has been performed in order to obtain new long-acting diuretics. The aim of this study was to decrease the metabolism of the drug and to slow down its rate of excretion by increasing its hydrophobicity. The present study describes the synthesis and the inhibitory potency of new torasemide derivatives in the bioassay system of the cortical thick ascending limb of rabbit. A correlation between the lipophilicity (log P') of these substances and their activity as inhibitors of the Na+ Cl- K+ co-transporter was observed. The present design led to compounds more active than torasemide. Structure-activity relationships permit us to propose an interaction model between torasemide derivatives and the Na+ 2Cl- K+ co-transport system of the cortical thick ascending limb.

Reduced phosphate transport in the renal proximal tubule cells in cystinosis is due to decreased expression of transporters rather than an energy defect

Nephropathic cystinosis is an autosomal recessive disorder caused by mutations in the CTNS gene [1], which encodes for a transporter (cystinosin) responsible for cystine efflux from lysosomes. In cystinotic renal proximal tubules (RPTs), the defect in cystinosin function results in reduced reabsorption of solutes by apical Na +/solute cotransport systems, including the Na +/phosphate (Pi) cotransport system [2]. However the underlying molecular mechanisms are unknown, given the lack of an appropriate cellular model. To obtain such a model system, we have knocked down cystinosin with siRNA in primary RPT cell cultures. An 80% reduction in cystinosin strongly inhibited Na + dependent Pi uptake (70%). Although this finding could be explained by a direct effect on transporters as well as by altered energetics (the ATP level dropped by 52%), our results demonstrate a lack of involvement of Na, K-ATPase, and a reduction in the number of NaPi2a transporters.

Clinical consequences of the biphasic elimination kinetics for the diuretic effect of furosemide and its acyl glucuronide in humans.

This review discusses the possibility of whether furosemide acyl glucuronide, a metabolite of furosemide, contributes to the clinical effect of diuresis. First an analytical method (e.g. HPLC) must be available to measure both parent drug and furosemide acyl glucuronide. Then, with correctly treated plasma and urine samples (light protected, pH 5) from volunteers and furosemide-treated patients, the kinetic curves of both furosemide as well as its acyl glucuronide can be measured. The acyl glucuronide is formed in part by the kidney tubules and it is possible that the compound is pharmacologically active through inhibition of the Na+/2Cl-/K+ co-transport system; up to now the mechanism of action has been solely attributed to furosemide. The total body clearance of furosemide occurs by hepatic and renal glucuronidation (50%) and by renal excretion (50%). Enterohepatic cycling of furosemide acyl glucuronide, followed by hydrolysis, results in a second and slow elimination phase with a half-life of 20-30 h. This slow elimination phase coincides with a pharmacodynamic rebound phase of urine retention. After each dosage of furosemide, there is first a short stimulation of urine flow (4 h), which is followed by a 3-day recovery period of the body. The following clinical implications arise from study of the elimination kinetics of furosemide. Repetitive dosing must result in accumulation of the recovery period. Accumulation of furosemide and its acyl glucuronide in patients with end-stage renal failure results from infinite hepatic cycling. Impaired kidney function may result in impaired glucuronidation and diuresis. While kidney impairment normally requires a dose reduction for those compounds which are mainly eliminated by renal excretion, for diuretics, a dose increment is required in order to maintain a required level of diuresis. The full clinical impact of the accumulation of furosemide and its acyl glucuronide in patients with end-stage renal failure has to be determined.

Tracing chameleon: thallium-201 SPECT/CT for pheochromocytoma.

A 61-year-old woman, in receipt of haemodialysis for 11 years, was diagnosed with a high-grade papillary urothelial carcinoma of the urinary bladder. Thallium-201 single photon emission computed tomography/computed tomography (SPECT/CT) was performed during the cancer work-up (Figure ​(Figure1).1). Unenhanced CT images of the SPECT/CT demonstrated a left adrenal mass (31.7 mm in size) with a homogeneous density of 62 HU. Scintigraphic SPECT images of the SPECT/CT showed an abnormal focus of increased radiotracer activity in the left upper quadrant of abdomen. The corresponding SPECT/CT fusion images confirmed the focus in the left adrenal medulla. The patient underwent a left adrenalectomy and the pathological findings indicated the histological characteristics of pheochromocytoma (Figure ​(Figure22). Fig. 1 New advance of image-rendering display: thallium-201 SPECT/CT. The coronal (left column), sagittal (middle column) and transaxial (right column) views of the CT images (top row) and corresponding SPECT images (middle row) fused to the SPECT/CT images ... Fig. 2 A pathological section of the left adrenal gland revealed a uniform proliferation of medullary cells, showing a nesting (Zellballen) pattern composed of well-defined cell clusters with amphophilic cytoplasm surrounded by fibrovascular stroma (haematoxylin ... Pheochromocytoma is a rare catecholamine-secreting tumour with a potential for cardiovascular devastation if the diagnosis is overlooked. The tumour may present with a diversity of imaging features that result from various histological degenerations including fibrosis, calcification, congestion, haemorrhage, necrosis and cystic change. Therefore, pheochromocytoma is often referred to as an epithet ‘imaging chameleon’ [1]. An adrenal mass with attenuation value >10 HU on CT usually suggests a radiological diagnosis of pheochromocytoma. Thallium-201, a widely used radiotracer for myocardial perfusion scintigraphy, has been proved effective in the diagnosis of various tumours through the mechanisms of increased vascularity and higher mitotic rate in tumour cells. Its uptake into tumour cells also reflects the cell membrane functions of the Na+/K+ ATPase pump, Tl+-Na+-2Cl− cotransport system and calcium-dependent ion channel. We firstly report a long-term haemodialysis patient whose pheochromocytoma is visualized through thallium-201 uptake within the left adrenal medulla by means of real-time functional SPECT and anatomical CT fusion images in tracing the site of pheochromocytoma. Diagnosis of pheochromocytoma in long-term haemodialysis patients is quite difficult since symptoms and signs implying pheochromocytoma are easily attributed to haemodialysis effects or complications of chronic renal disease. Measurement of urinary catecholamines and their metabolites is not feasible in anuric patients. Plasma catecholamines are also unreliable due to the impact of haemodialysis. With the help of advanced imaging fusion science, integrated thallium-201 SPECT/CT could be a promising paradigm for the diagnosis of pheochromocytoma in haemodialysis patients [2]. Conflict of interest statement. None declared.

Counterpoint: Sickle cell trait should not be considered asymptomatic and as a benign condition during physical activity

Counterpoint: Sickle cell trait should not be considered asymptomatic and as a benign condition during physical activity

Physiopathologie de la diarrhée à rotavirus

The rotavirus is the major cause of infantile gastroenteritis. The virus infects the mature enterocytes of the villus tip of the small intestine and induces a watery diarrhea. Diarrhea can occur in the absence of histological changes in the intestine, and, conversely, the histological changes can be asymptomatic. Rotavirus decreases the activities of digestive enzymes at the apical brush border membrane and inhibits Na+ -solute cotransport systems. Accumulation of carbohydrates in the intestinal lumen as well as malabsorption of nutrients and a concomitant inhibition of water absorption can lead to a malabsorptive component of diarrhea. Since the discovery of the NSP4 enterotoxin, several hypotheses have been proposed in favour of an additional secretion component in the pathogenesis of diarrhea. Rotavirus induces a moderate net chloride secretion at the onset of the diarrhea. The mechanisms appear to different from those used by bacterial enterotoxin that cause pure secretory diarrhea. Rotavirus stimulated C1- reabsorption in villi, and failed to stimulate C1- secretion in crypt. Intestinal villi could secrete chloride as a result of rotavims infection. The chloride secretory response is regulated by a dependant calcium signalling pathway induced by NSP4. The overall response is weak, suggesting that NSP4 may exert both secretory and subsequent antisecretory actions, hence limiting C1- secretion.

Phosphate transport by proteoid roots of Hakea sericea

Up to now the higher capacity of proteoid roots to absorb inorganic phosphate from P i-poor soils has been related mainly to their increased root surface area and higher exudation of organic acids and phosphatases, while much less attention has been directed to their mechanisms of P i uptake. Here we report a characterization of the P i uptake kinetics of the proteoid root-forming species Hakea sericea Schrad. This Proteaceae is an Australian native, which is disseminating very fast through forests of the European south. Dense mats of proteoid roots were observed in the upper soil layers of the invaded area in Portugal where availability of P and N was shown to be very low. Plants grown hydroponically under low-P i also developed proteoid roots, and the proteoid clusters presented a major role in P i absorption in comparison to the non-proteoid portions of the root system as revealed by their higher 32P i labeling. The 32P i uptake by proteoid roots was dependent on H + gradient and yielded a biphasic kinetics, suggesting the involvement of H +/P i co-transport systems with K m values of 0.225 and 40.8 μM P i. The analogs phosphite (Phi) and arsenate, but not vanadate, inhibited competitively the P i absorption. Such biphasic P i uptake pattern with the highest affinity at submicromolar range is likely to be of critical importance for the capacity of this plant species to invade and proliferate throughout vast areas of nutrient-deprived soils.

Influence of deoxynivalenol on the D‐glucose transport across the isolated epithelium of different intestinal segments of laying hens

Deoxynivalenol (DON) decreases glucose absorption in the proximal jejunum of laying hens in vitro and this effect is apparently mediated by the inhibition of the sodium D-glucose co-transporter. DON could modulate the sugar transport of other intestinal regions of chickens. For this purpose, we have measured the effects of DON on the Na(+) D-glucose co-transporter, by addition of DON after and before a glucose addition in the isolated epithelium from chicken duodenum, jejunum, ileum, caecum and colon by using the Ussing chamber technique in the voltage clamp technique. The data showed in all segments of the gut that the addition of D-glucose on the mucosal side produced an increase in the current (Isc) compared with the basal values, the Isc after glucose addition to the small intestine was greater than the Isc of the large intestine compared with the basal values, specially of the jejunum (p < 0.002), indicating that the jejunum is the segment that is the best prepared for Na(+)-D-glucose co-transport. Further addition of 10 microg DON/ml to the mucosal solution decreased the Isc in all segments and the Isc returned to the basal value, especially in the duodenum and mid jejunum (p < 0.05). In contrast, the addition of 5 mmol D-glucose/l on the mucosal side after incubation of the tissues with DON in all segments had no effect on the Isc (p > 0.05), suggesting that DON previously inhibited the Na(+)D-glucose co-transport. The blocking effects of DON in duodenum and jejunum were greater than the other regions of the gut. It can be concluded that the small intestine of laying hens has the most relevant role in the carrier mediated glucose transport and the large intestine, having non-significant capacity to transport sugars, appears to offer a minor contribution to glucose transport because the surface area is small. The effect of D-glucose on the Isc was reversed by DON in all segments, especially in the duodenum and jejunum, suggesting that DON entirely inhibited Na(+)-D-glucose co-transport. This finding indicates that the inhibition of Na(+) co-transport system in all segments could be an important mode of action for DON toxicity of hens.

Non-Involvement of the Human Monocarboxylic Acid Transporter 1 (MCT1) in the Transport of Phenolic Acid

Phenolic acids such as p-coumaric acid and microbial metabolites of poorly absorbed polyphenols are absorbed by the monocarboxylic acid transporter (MCT)-mediated transport system which is identical to the fluorescein/H(+) cotransport system. We focus here on the physiological impact of MCT-mediated absorption and distribution. We examined whether MCT1, the best-characterized isoform found in almost all tissues, is involved in this MCT-mediated transport system. The induction of MCT1 expression in Caco-2 cells by a treatment with sodium butyrate (NaBut) did not increase the fluorescein permeability. Moreover, the transfection of Caco-2 cells with an expression vector encoding MCT1 caused no increase in either the permeability or uptake of fluorescein. Furthermore, in the MCT1-expressing oocytes, no increase of p-coumaric acid uptake was apparent, whereas the uptake of salicylic acid, a substrate of MCT1, nearly doubled. Our data therefore establish that MCT1 was not involved in the MCT-mediated transport of phenolic acids.

Characterization of salicylate uptake across the basolateral membrane of the Malpighian tubules of Drosophila melanogaster

The organic anion salicylate is a plant secondary metabolite that can protect plants against herbivores. Transport of salicylate across the basolateral membrane of the Malpighian tubules of Drosophila melanogaster was studied using a radioisotope tracer technique. The uptake of [ 14C]salicylate by the Malpighian tubules was active, saturable and Na +-dependent; the maximum uptake rate ( J max) and the half saturation concentration ( K t) were 12.6 pmol tubule −1 min −1 and 30.7 μmol l −1, respectively. In contrast to organic anion transport by vertebrate renal tissues, salicylate uptake was not trans-stimulated by glutarate (0.01–1.0 mmol l −1) or cis-inhibited by high concentrations (5 mmol l −1) of various α-keto acids (glutaric acid, α-ketoglutaric acid, succinic acid, and citric acid). Changes in basolateral membrane potential or physiologically relevant changes in bathing saline pH did not affect the rate of [ 14C]salicylate uptake. Ring-structure monocarboxylic acids (benzoic acid, nicotinic acid, gentisic acid, unlabelled salicylic acid, α-cyano-4-hydroxycinnamic acid, probenecid, fluorescein, and P-aminohippuric acid) strongly inhibited [ 14C]salicylate uptake rate. In contrast, short-chain monocarboxylic acids had little (butyric acid) or no effect (lactic acid, pyruvic acid, and propionic acid). Our results suggest that salicylate uptake across the basolateral membrane of D. melanogaster Malpighian tubules is mediated by a non-electrogenic, α-cyano-4-hydroxycinnamic acid-sensitive, Na +:salicylate cotransport system.

Effects of MRP2-GSH cotransport system on hepatic arsenic metabolism in rats

To investigate the role of multidrug resistant protein 2 (MRP2) and glutathione (GSH) cotransport system in hepatic arsenic metabolism in rats. Thirty healthy Wistar rats were divided randomizedly into five groups. The first group was the control group and the rats in this group were administered with normal saline. In the second, third and fourth group the rats were administered with 4, 10 and 20 mg As(+)3/kg BW of sodium arsenite respectively every other day for two weeks. The fifth group was the benzene-soluble organics (BSO) intervention group and in this group the rats were administered with 2 mmol/kg BW BSO intraperitoneally every day three days before the end of the experiment. The other treatment was the same as in other groups. All rats were sacrificed two weeks after the treatments. Arsenic contents in bile, liver and blood were detected by atomic absorption spectroscopy (AAS), and the expression of MRP2 in the membrane of hepatocyte was determined by Western-blot analysis. The level of total arsenic (including organic arsenic and inorganic arsenic) in bile, liver and blood in all three different dose groups was higher than those in the control groups (P < 0.05). Arsenic levels of bile and liver were increased with intragastric arsenic dose. Blood arsenic levels were not significantly different in three different dose groups. Expression of hepatic MRP2 was increased with intragastric arsenic concentration. A positive correlation between biliary arsenic concentration and MRP2 levels was found in liver (r = 0.986, P < 0.05). For the rats pretreated with BSO, the biliary arsenic was significantly higher than that in the control group but lower than that in the high dose group; the liver and blood arsenic was higher than that in the control group and in the high dose group. Expression of MRP2 pretreated with BSO was decreased. Sodium arsenite can induce expression of MRP2 and the up-regulation of MRP2 may play an important role in the bile secretion of arsenite and its metabolites. The function of MRP2 for transportation of arsenic and its metabolites is associated with the intracellular GSH level. BSO inhibits the synthesis of GSH, which weakens the function of the MRP2-GSH cotransport system and makes the liver arsenic increased.

Purification by p-aminobenzoic acid (PABA)-affinity chromatography and the functional reconstitution of the nateglinide/H + cotransport system in the rat intestinal brush-border membrane

(−)- N-( trans-4-Isopropylcyclohexanecarbonyl)- d-phenylalanine (nateglinide) is a novel oral hypoglycemic agent possessing a peptide-type bond and a carboxyl group in its structure. Recently, we have shown that nateglinide transport occurs via the ceftibuten/H + cotransport system, which is distinct from PepT1, and that the fluorescein/H + cotransport system is involved in the uptake of nateglinide. The aim of this study was to characterize the functional properties of the intestinal nateglinide transporter. In the first part of this study, we demonstrated that the ceftibuten/H + cotransport system is identical to the fluorescein/H + cotransport system. We succeeded in purification of the nateglinide transporter from brush-border membranes of the rat small intestine using p-aminobenzoic acid (PABA)-affinity chromatography. We then investigated the functional properties of the nateglinide transporter using proteoliposomes prepared from the PABA-affinity chromatography elute. We demonstrated that nateglinide, ceftibuten, and fluorescein are transported by the same transporter in the intestine.

Isophosphoramide Mustard-Lysine (ZIO-201): Active Moiety of Ifosfamide (IFOS) without Kidney Toxicity.

Ifosfamide (IFOS) and cyclophosphamide (CPA) are widely used anti-cancer drugs. Both are pro-drugs which must be metabolized before their metabolites cross-link DNA. Use of these drugs, especially high-dose IFOS, is complicated by substantial adverse effects of metabolites not directly involved in DNA-cross-linking including kidney, bladder and CNS toxicities. Recently, a lysine-stabilized form of isophosphoramide mustard (IPM-lysine; ZIO-201), an IFOS-metabolite, was developed. ZIO-201 directly cross-links DNA and is active against human cancer cell lines and in mice with human cancer xenografts. To evaluate nephrotoxicity of ZIO-201 we used an in vitro primary rabbit kidney proximal tubule (RPT) cell culture system, which retains many of the characteristics of renal proximal tubule cells including a polarized morphology, a Na+/glucose cotransport system and a p-aminohippurate transport system, glutathione status, and hormone responses (including a parathyroid hormone sensitive adenylate cyclase). We studied effects of ZIO-201 and other IFOS metabolites on primary RPT cells. IFOS is activated by the cytochrome P450 system producing a mixture of 2- and 3-dechloroethylifosfamide and resulting in formation of chloroacetaldehyde (CAA). Also, metabolism of IFOS by the cytochrome P450 system produces 4-hydroxyifosfamide, ultimately resulting in the production of acrolein (ACR). CAA and ACR are IFOS metabolites implicated in kidney toxicity in humans. Effects of ZIO-201 and CAA on the viability of confluent monolayers of primary rabbit kidney proximal tubule (RPT) cells were tested at 15–100 μM. Viability was determined by neutral red dye uptake by the primary cultures after 8 h incubation. ZIO-201 did not significantly reduce viability of primary RPT cells; the LD50 for CAA was about 40 μM with complete inhibition at 75 μM. Effects of 4-hydroperoxyifosfamide (HIFA), a synthetic prodrug of 4-hydroxyifosfamide and ACR were also studied. Although ACR had an LD50 of 80 μM HIFA did not significantly affect viability. The IFOS metabolite 4-hydroxyifosfamide, was prepared from 4-hydroperoxyifosfamide (1). At 200 μM, 4-hydroxyifosfamide resulted in extensive cell death after 4 h incubation. These data show that ZIO-201 avoids the kidney toxicity caused by metabolites of pro-drugs like IFOS. Whereas 2 IFOS metabolites, CAA and ACR were cytotoxic to kidney cells, ZIO-201 was not. An explanation for the toxic effects of 200 μM 4-hydroxyifosfamide t might be metabolism to ACR in vitro.