Brush Border Membrane Transport Research Articles

Intestinal Epithelial Cell Brush Border Membrane Cl:HCO3 Exchanger Regulation by Mast Cells in Chronic Ileitis.

Malabsorption of NaCl is the primary cause of diarrhea in inflammatory bowel disease (IBD). Coupled NaCl absorption occurs via the dual operation of Na:H and Cl:HCO3 exchange in the brush border membrane (BBM) of villus cells. Cl:HCO3 exchange is mediated by BBM transporters DRA (downregulated in adenoma) and PAT1 (putative anion transporter 1) in the mammalian small intestine. DRA/PAT1-mediated Cl:HCO3 exchange was significantly downregulated in the BBM of villus cells in a rabbit model of chronic ileitis, while Na:H exchange was unaffected. The inhibition of Cl:HCO3 exchange was restored in the rabbits when treated with a broad-spectrum immunomodulator, i.e. a glucocorticoid, indicating that the downregulation of DRA/PAT1 is likely to be immune-mediated during chronic enteritis. Mucosal mast cells are one type of key immune cells that are known to proliferate and release immune inflammatory mediators, thus playing a significant role in the pathogenesis of IBD. However, how mast cells may regulate DRA- and PAT1-mediated Cl:HCO3 exchange in a rabbit model of chronic ileitis is unknown. In this study, treatment of rabbits with chronic intestinal inflammation with the mast cell stabilizer ketotifen did not affect the mucosal architecture of the inflamed intestine. However, ketotifen treatment reversed the inhibition of Cl:HCO3 activity in the BBM of villus cells. This restoration of Cl:HCO3 activity to normal levels by ketotifen was found to be secondary to restoring the affinity of the exchangers for its substrate chloride. This observation was consistent with molecular studies, where the mRNA and BBM protein expressions of DRA and PAT1 remained unaffected in the villus cells under all experimental conditions. Thus, this study indicates that mast cells mediated the inhibition of coupled NaCl absorption by inhibiting Cl:HCO3 exchange in a rabbit model of chronic enteritis.

Dietary Tryptophan‐Lactobacillus rhamnosus GG Interactions Modify Intestinal Differentiation and Function

Although the influence of the gut microbiota on structure and proliferation of intestinal epithelial cells has been established, there is disconcordance on information related to cell type differentiation, because most studies on probiotics reflected an outcome from interactions among Lactobacillus, numerous existing microbes, and the host, with limited insight on the specific impact of Lactobacilli spp. For example, L. reuteri enhanced goblet and Paneth cell differentiation in intestine of conventionally‐raised chickens with an established commensal gut microbiota, confounding results. Here we fed gnotobiotic mice with tryptophan (t+) or trp‐free (t–) diets, followed by monocolonization with LGG (L+, ATCC 53103) or without (control, L–) for a total of 4 groups (L+t+, L+t–, L–t+, L–,t–), then ileum transcriptome was analyzed 3 wk later. L– mice remained germ‐free throughout while only LGG was associated with L+. Principal component analysis shows L+ or t+ by itself to modestly cluster away from L–t–, but the cluster depicting the combination of L+t+ was far removed from the others. The heatmap of whole ileum transcriptome shows several distinct groups of genes affected by t+ or by L+ alone. However, the simultaneous impact of L+t+ dramatically upregulates numerous gene clusters while downregulating a few. Volcano plots and Venn diagrams comparing L–t– with L–t+, or L+t– with L–t– indicate ~2300 unique genes affected by L+ (LGG) or t+ (tryptophan) alone. Comparisons between L+t+ and L+t– (effect of t with L) or between L+t+ and L–t+ (effect of L with t) each impact >3800 genes, suggesting that synergistic interactions between L+ and t+ primarily underlie the probiotic impact ascribed to LGG and dietary t. Many of the genes upregulated by L+t+ have functions typical of mature enterocytes: tight junctions, brush border membrane transporters and enzymes, intestinal bile acid metabolism, lipid synthesis and fatty acid b‐oxidation. Many downregulated genes are antimicrobial and secreted peptides typical of differentiated Paneth and goblet cells. This latter result was recapitulated when reexamined in conventional mice perfused for 4 h with LGG, as expression of many defensins decreased by >10‐fold, suggesting not only that this probiotic’s effects can arise from both acute ex vivo and chronic in vivo interactions with the host, but also in hosts housing a complex microbiota. Remarkably, Notch ligands Dll1 and Dll4 typically expressed in Paneth cells as well as Yap1, Axin2 and Ctnnb1 are upregulated in L+t+ ileum, suggesting downstream upregulation of Hes1 and repression of Atoh1, leading to suppression of the secretory lineage. In conclusion, LGG monocolonization may lead to synthesis of t+‐derived metabolites by both host and bacteria, leading to an enhancement of absorptive enterocyte functions and suppression of those by goblet and Paneth cells.

Stimulation of constitutive nitric oxide uniquely and compensatorily regulates intestinal epithelial cell brush border membrane Na absorption.

In the mammalian small intestine, sodium is primarily absorbed by Na+/H+ exchange (NHE3) and Na‐glucose cotransport (SGLT1) in the brush border membrane (BBM) of villus cells. However, how enhanced cellular constitutive nitric oxide (cNO) may affect NHE3 and SGLT1 remains unclear. Both in vivo in rabbit intestinal villus cells and in vitro IEC‐18 cells, administration of NO donor, GSNAP, modestly increased cNO. GSNAP stimulated SGLT1 in villus and IEC‐18 cells. The mechanism of stimulation was secondary to an increase in the affinity of SGLT1 for glucose. The change in SGLT1 was not secondary to altered Na‐extruding capacity of the cell since Na+/K+‐ATPase was decreased by GSNAP treatment. In contrast, GSNAP inhibited NHE3 activity in villus cell BBM. The mechanism of NHE3 inhibition was secondary to reduced BBM transporter numbers. These studies demonstrated that the physiological increase in cNO uniquely regulates mammalian small intestinal NHE3 and SGLT1 to maintain Na homeostasis.

Expression of small intestinal nutrient transporters in embryonic and posthatch turkeys

Nutrients are absorbed in the small intestine through a variety of transporter proteins, which have not been as well characterized in turkeys as in chickens. The objective of this study was to profile the mRNA expression of amino acid and monosaccharide transporters in the small intestine of male and female turkeys. Jejunum was collected during embryonic development (embryonic d 21 and 24, and d of hatch (DOH)) and duodenum, jejunum, and ileum were collected in a separate experiment during posthatch development (DOH, d 7, 14, 21, and 28). Real-time PCR was used to determine expression of aminopeptidase N (APN), one peptide (PepT1), 6 amino acid (ASCT1, b(o,+)AT, CAT1, EAAT3, LAT1, y(+)LAT2) and 3 monosaccharide (GLUT2, GLUT5, SGLT1) transporters. Data were analyzed by ANOVA using JMP Pro 11.0. APN, b(o,+)AT, PepT1, y(+)LAT2, GLUT5, and SGLT1 showed increased expression from embryonic d 21 and 24 to DOH. During posthatch, all genes except GLUT2 and SGLT1 were expressed greater in females than males. GLUT2 was expressed the same in males as females and SGLT1 was expressed greater in males than females. All basolateral membrane transporters were expressed greater during early development then decreased with age, while the brush border membrane transporters EAAT3, GLUT5, and SGLT1 showed increased expression later in development. Because turkeys showed high-level expression of the anionic amino acid transporter EAAT3, a direct comparison of tissue-specific expression of EAAT3 between chicken and turkey was conducted. The anionic amino acid transporter EAAT3 showed 6-fold greater expression in the ileum of turkeys at d 14 compared to chickens. This new knowledge can be used not only to better formulate turkey diets to accommodate increased glutamate transport, but also to optimize nutrition for both sexes.

Intestinal absorption of cephalosporin antibiotics: correlation between intestinal absorption and brush-border membrane transport.

The absorption of seven cephalosporin antibiotics from the in-situ intestinal loop at pH 7.4 and their transport by brush-border membrane vesicles in the presence of an inward H+ gradient ([pH]i = 7.5, [pH]o = 6.0) were examined. A good correlation was found between the intestinal absorption rate and the initial uptake rate by brush-border membrane vesicles. The data suggest that the transport study using intestinal brush-border membrane vesicles is useful as a model system for the intestinal absorption of beta-lactam antibiotics.

Peptide transport and animal growth: the fish paradigm

Protein digestion products are transported from the intestinal lumen into the enterocyte both in the form of free amino acids (AAs), by a large variety of brush border membrane AA transporters, and in the form of di/tripeptides, by a single brush border membrane transporter known as PEPtide Transporter 1 (PEPT1). Recent data indicate that, at least in teleost fish, PEPT1 plays a significant role in animal growth by operating, at the gastrointestinal level, as part of an integrated response network to food availability that directly supports body weight. Notably, PEPT1 responds to both fasting and refeeding and is involved in a phenomenon known as compensatory growth (a phase of accelerated growth when food levels are restored after a period of growth depression). In particular, PEPT1 expression decreases during fasting and increases during refeeding, which is the opposite of what observed so far in mammals and birds. These findings in teleost fish document, to our knowledge, for the first time in a vertebrate model, a direct correlation between the expression of an intestinal transporter, such as PEPT1, primarily involved in the uptake of dietary protein degradation products and animal growth.

Studies on the protective effect of dietary fish oil on uranyl-nitrate-induced nephrotoxicity and oxidative damage in rat kidney

Human and animal exposure demonstrates that uranium is nephrotoxic. However, attempts to reduce it were not found suitable for clinical use. Dietary fish oil (FO) enriched in omega-3 fatty acids reduces the severity of cardiovascular and renal diseases. Present study investigates the protective effect of FO on uranyl nitrate (UN)-induced renal damage. Rats prefed with experimental diets for 15 days, given single nephrotoxic dose of UN (0.5mg/kg body weight) intraperitoneally. After 5d of UN treatment, serum/urine parameters, enzymes of carbohydrate metabolism, brush border membrane (BBM), oxidative stress and phosphate transport were analyzed in rat kidney. UN nephrotoxicity was characterized by increased serum creatinine and blood urea nitrogen. UN increased the activity of lactate dehydrogenase and NADP-malic enzyme whereas decreased malate, isocitrate and glucose-6-phophate dehydrogenases; glucose-6-phophatase, fructose-1, 6-bisphosphatase and BBM enzyme activities. UN caused oxidant/antioxidant imbalances as reflected by increased lipid peroxidation, activities of superoxide dismutase, glutathione peroxidase and decreased catalase activity. Feeding FO alone increased activities of enzymes of glucose metabolism, BBM, oxidative stress and Pi transport. UN-elicited alterations were prevented by FO feeding. However, corn oil had no such effects and was not similarly effective. In conclusion, FO appears to protect against UN-induced nephrotoxicity by improving energy metabolism and antioxidant defense mechanism.

Studies on the protective effect of green tea against cisplatin induced nephrotoxicity

Cisplatin (CP) an anticancer drug is known to induce nephrotoxicity, which limits its long-term clinical use. Green tea (GT), consumed since ancient times is known for its numerous health benefits. It has been shown to improve kidney functions in animal models of acute renal failure. The present study was undertaken to see whether GT can prevent CP-induced nephrotoxic and other deleterious effects. A nephrotoxic dose of CP was co-administered to control and GT-fed male Wistar rats every fifth day for 25 days. The effect of GT was determined on CP-induced alterations in various serum parameters and on enzymes of carbohydrate metabolism, brush border membrane, and antioxidant defense system in renal cortex and medulla. CP nephrotoxicity was recorded by increased serum creatinine and blood urea nitrogen. CP increased the activities of lactate dehydrogenase and acid phosphatase whereas, the activities of malate dehydrogenase, glucose-6-phosphatase, superoxide dismutase, catalase, and (32)Pi transport significantly decreased. GT consumption increased the activities of the enzymes of carbohydrate metabolism, brush border membrane, oxidative stress, and (32)Pi transport. GT ameliorated CP-induced nephrotoxic and other deleterious effects due to its intrinsic biochemical/antioxidant properties.

Protective effect of green tea extract on gentamicin-induced nephrotoxicity and oxidative damage in rat kidney

Gentamicin (GM) is an effective aminoglycoside antibiotic against severe infections but nephrotoxicity and oxidative damage limits its long term clinical use. Various strategies were attempted to ameliorate GM nephropathy but were not found suitable for clinical practice. Green tea (GT) polyphenols have shown strong chemopreventive and chemotherapeutic effects against various pathologies. We hypothesized that GT prevents GM nephrotoxicity by virtue of its antioxidative properties. A nephrotoxic dose of GM was co-administered to control and GT-fed male Wistar rats. Serum parameters and enzymes of oxidative stress, brush border membrane (BBM), and carbohydrate metabolism were analyzed. GM increased serum creatinine, cholesterol, blood urea nitrogen (BUN), lipid peroxidation (LPO) and suppressed superoxide dismutase (SOD) and catalase activities in renal tissues. Activity of hexokinase, lactate dehydrogenase increased whereas malate dehydrogenase decreased. Gluconeogenic enzymes and glucose-6-phosphate dehydrogenase were differentially altered in the cortex and medulla. However, GT given to GM rats reduced nephrotoxicity parameters, enhanced antioxidant defense and energy metabolism. The activity of BBM enzymes and transport of Pi declined by GM whereas GT enhanced BBM enzymes and Pi transport. In conclusion, green tea ameliorates GM elicited nephrotoxicity and oxidative damage by improving antioxidant defense, tissue integrity and energy metabolism.

Studies on the protective effect of dietary fish oil on gentamicin-induced nephrotoxicity and oxidative damage in rat kidney

Gentamicin (GM)-induced nephrotoxicity limits its long-term clinical use. Several agents/strategies were attempted to prevent GM nephrotoxicity but were not found suitable for clinical practice. Dietary fish oil (FO) retard the progression of certain types of cancers, cardiovascular and renal disorders. We aimed to evaluate protective effect of FO on GM-induced renal proximal tubular damage. The rats were pre-fed experimental diets for 10 days and then received GM (80 mg/kg body weight/day) treatment for 10 days while still on diet. Serum/urine parameters, enzymes of carbohydrate metabolism, brush border membrane (BBM), oxidative stress and phosphate transport in rat kidney were analyzed. GM nephrotoxicity was recorded by increased serum creatinine and blood urea nitrogen. GM increased the activities of lactate and glucose-6-phosphate dehydrogenases whereas decreased malate, isocitrate dehydrogenases; glucose-6 and fructose-1,6-bisphosphatases; superoxide dismutase, catalase, glutathione peroxidase and BBM enzymes. In contrast, FO alone increased enzyme activities of carbohydrate metabolism, BBM and oxidative stress. FO feeding to GM treated rats markedly enhanced resistance to GM elicited deleterious effects and prevented GM-induced decrease in 32Pi uptake across BBM. Dietary FO supplementation ameliorated GM-induced specific metabolic alterations and oxidative damage due to its intrinsic biochemical/antioxidant properties.

NPC1L1 and SR‐BI are Involved in Intestinal Cholesterol Absorption from Small‐Size Lipid Donors

In the human intestinal content after a meal, cholesterol is dispersed in a complex mixture of emulsified droplets, vesicles, mixed micelles and precipitated material. The aim of this study was to determine the contribution of the main intestinal cholesterol transporters (NPC1L1, SR-BI) to the absorption processes, using different cholesterol-solubilizing donors. Cholesterol donors prepared with different taurocholate concentrations were added to an apical medium of differentiated TC7/Caco-2 cells. As the taurocholate concentrations increased, cholesterol donor size decreased (from 712 to 7 nm in diameter), which enhanced cholesterol absorption in a dose-dependent manner (38-fold). Two transport processes were observed: (1) absorption from large donors exhibited low-capacity transport with no noticeable transporter contribution; (2) efficient cholesterol absorption occurs from small lipid donors (<or=23 nm diameter), mainly due to NPC1L1 and SR-BI involvement. In addition, bile acids significantly increased mRNA and protein expression of NPC1L1, but not of SR-BI. In conclusion, bile acids present in the intestinal lumen and the micelles enhance intestinal cholesterol transport into the cell by two different regulatory processes: by reducing the lipid donor size, so that small-size mixed micelles can more easily access brush-border membrane transporters, and by increasing the expression level of the enterocyte NPC1L1. These mechanisms could account for the important inter-individual variations observed in cholesterol intestinal absorption.

Effects of Angiotensin II Receptor Blockers on Renal Handling of Uric Acid in Rats

Elevated serum uric acid level has been associated with increased cardiovascular risk in hypertensive patients. Several angiotensin II receptor blockers exhibit differential effects on regulation of serum uric acid level in humans. We have demonstrated that some angiotensin II receptor blockers trans-stimulate the uptake of uric acid by human URAT1 and others inhibit the transport of uric acid mediated by human URAT1, OAT1, OAT3 and MRP4 in vitro. This study investigated the effects of candesartan, pratosartan and telmisartan on renal handling of uric acid in rats in vivo and in vitro. Candesartan (0.1 mg/kg) significantly decreased the urinary excretion of uric acid and increased the plasma uric acid concentration. The kidney candesartan level after low-dose treatment is close to that required to trans-stimulate uric acid uptake in vitro. Pratosartan exhibited dose-dependent hypouricemic and uricosuric effects, while telmisartan showed no effects on plasma uric acid level. Furthermore, we confirmed the effects of the tested drugs on uric acid transport by rat renal brush border membrane transporter(s) and basolateral Oat1 and Oat3. Effects of angiotensin II receptor blockers in rats may be mainly determined by their intrinsic effects (cis-inhibition and trans-stimulation) on uric acid reabsorption transporter(s) and their pharmacokinetic properties in rats.

Novel insights into intestinal and renal folate transport. Focus on “Apical membrane targeting and trafficking of the human proton-coupled folate transporter in polarized epithelia”

FOLATE IS AN ESSENTIAL MICRONUTRIENT that functions as a coenzyme in DNA and RNA synthesis and in the metabolism of several amino acids, including homocysteine (11). Folate deficiency can occur in humans because of poor dietary intake, malabsorption, metabolic blocks, or increased requirements as seen during pregnancy and lactation (3). Symptoms of deficiency are severe and include megoblastic anemia and neural tube defects in children. Mammals must absorb folate from the diet, because they do not have the capability to synthesize folate. Intestinal absorption occurs by both passive and carriermediated mechanisms, with the second process predominating in the proximal small intestine at normal intake levels (3). Intestinal transport is critical for overall body folate homeostasis, as exemplified by the fact that individuals with hereditary folate malabsorption exhibit signs of folate deficiency (5, 6). Dietary folate exists in the polyglutamate form, which is converted to the monoglutamate form before absorption. Transport across the intestinal epithelium is a two-step process; the putative brush-border membrane transporter is pH dependent (i.e., higher activity at low pH) (12), and movement across the basolateral surface is also a carrier-mediated process that has been previously described (13). Recently, three folate transport systems have been identified in mammals: the reduced folate carrier (RFC) (4, 8), the folate receptor (FR) (7), and the newly described proton-coupled folate transporter (PCFT; also called SLC46A1) (9). The relative role of each of these transport systems in intestinal and renal folate absorption is currently not completely understood (although the FR, which was described in renal cells, has not been identified in the intestine). The recent studies by Subramanian et al. (17) have provided novel evidence that makes a major contribution to our understanding of folate homeostasis. These authors have clearly demonstrated, for the first time, that the human PCFT is a brush-border membrane protein in polarized epithelia; they show that it is functionally expressed on the apical membrane in widely accepted models of the intestinal and renal epithelia. Moreover, in polarized MDCK cells, they noted that the PCFT and the RFC showed differential localization, with the RFC present on the basolateral domain and the PCFT in the apical membrane. Additional experiments were aimed at determining the trafficking motifs of the PCFT and the effect of clinically relevant mutations; results showed that the COOH terminus was not important for apical targeting and that mutation of a region (DXXGR[113]R) associated with hereditary folate malabsorption (the R[113]S mutation has been identified in a human patient) (20) abolished a -turn between membrane-spanning domains 2 and 3 and led to protein retention in the endoplasmic reticulum. Further analyses found that cell surface delivery of the PCFT is dependent on an intact microtubular network. The findings presented in this article reveal new aspects of folate transport that can only be appreciated in the context of previous studies in this field. Prior investigations suggested that the RFC is important in intestinal and renal folate absorption. Some studies found RFC expressed on the intestinal brush-border membrane in mouse (18) and rat (1) intestine, where it is presumably involved in folate absorption, while others described expression at the basolateral surface of renal epithelial cells (2) and at the plasma membrane when expressed in Xenopus oocytes (16). Moreover, the RFC was found to be ontogenically regulated during rat development and was reflective of overall intestinal folate transport (1). Despite these intriguing observations, certain inconsistencies exist between the known properties of intestinal folate transport and those ascribed to the RFC, including the higher activity of the RFC at neutral as opposed to acidic pH (15).

Vitamin C attenuates cisplatin-induced alterations in renal brush border membrane enzymes and phosphate transport

Cisplatin is a widely used antineoplastic agent that exhibits dose limiting nephrotoxicity. We have previously shown that the administration of cisplatin results in decrease in the activities of renal brush border membrane (BBM) enzymes and transport of inorganic phosphate (Pi) across BBM vesicles. In the present study we have investigated the effect of pre-treatment with vitamin C (ascorbic acid) on cisplatin-induced nephrotoxicity and changes in BBM enzymes and Pi transport. Administration of a single dose of cisplatin (6 mg/kg body weight) caused nephrotoxicity in rats that manifested biochemically as an elevation of serum urea nitrogen and creatinine levels. Treatment of rats with a single dose of vitamin C, six hours prior to administration of cisplatin, protected the kidney from the damaging effect of cisplatin. Vitamin C pre-treatment significantly decreased the urea nitrogen and creatinine levels. It attenuated the cisplatin-induced reduction in the activities of BBM and anti-oxidant enzymes and also Pi transport. These results suggest that vitamin C is an effective chemoprotectant against cisplatin-induced acute renal failure and dysfunction of the renal BBM in rats.

The transport of glutamine into mammalian cells

Glutamine has many important functions in mammalian cells, and glutamine transport across cell membranes has accordingly been extensively studied. In the past few years a number of important glutamine transport proteins have been sequenced and their molecular properties have been characterised. In general, four major transporters are important physiologically. These are known as (i) SNAT3 (System N) which is important in glutamine uptake in periportal cells in liver and in across the basolateral membrane of renal proximal tubule cells and is also involved in glutamine release by liver perivenous cells and by astrocytes; a variant of this protein catalyses glutamine release from skeletal muscle. (ii) SNAT1 (a specific System A sub-type) which is important in glutamine uptake by neuronal cells (iii) ASCT2 which is essential for glutamine uptake by rapidly growing epithelial cells and tumour cells in culture and (iv) the recently discovered brush border membrane transporter B0 AT1 (SLC6A19). Recent studies considered both the importance of ASCT2 in tumour cell growth and the regulation of ASCT2 expression. In SK-Hep hepatoma cells, knockdown of ASCT2 using antisense mRNA has been shown to cause apoptosis. Expression of the ASCT2 transporter in HepG2 hepatoma cells is stimulated by glutamine by a pathway involving the promoter element AGGTGAATGACTT which binds FXR/RXR dimers.

Arachidonic acid cascade and epithelial barrier function during Caco-2 cell differentiation

The small intestinal epithelium is a highly dynamic system continuously renewed by a process involving cell proliferation and differentiation. The intestinal epithelium constitutes a permeability barrier regulating the vectorial transport of ions, water, and solutes. Morphological changes during cell differentiation, as well as changes in the activity of brush-border enzymes and the expression of transport proteins, are well established. However, little is known about the arachidonic acid (AA) cascade underlying epithelial cell differentiation or its role in the development of epithelial barrier function. The main purpose of this study was to examine the activity of the high-molecular-weight phospholipases A(2) (PLA(2)) and cyclooxygenase (COX) pathway during differentiation, with particular emphasis on paracellular permeability. PLA(2) activity, AA release, COX-2 expression, prostaglandin E(2) (PGE(2)) production, and paracellular permeability were studied in preconfluent, confluent, and differentiated Caco-2 cell cultures. Our results show that Caco-2 differentiation induces a decrease in both calcium-independent PLA(2) activity and COX-2 expression and, consequently, a decrease in AA release and PGE(2) synthesis in parallel with a reduction in paracellular permeability. Moreover, the addition of PGE(2) to differentiated cells, at concentrations similar to those detected in nondifferentiated cultures, induces the disruption of epithelial barrier function. These results suggest that AA release by calcium-independent PLA(2), COX-2 expression, and subsequent PGE(2) release are important for the maintenance of paracellular permeability in differentiated Caco-2 cells.

Regulation of sodium-proton exchanger isoform 3 (NHE3) by PKA and exchange protein directly activated by cAMP (EPAC)

The Na(+)/H(+) exchanger 3 (NHE3) is expressed in the brush border membrane (BBM) of proximal tubules (PT). Its activity is down-regulated on increases in intracellular cAMP levels. The aim of this study was to investigate the contribution of the protein kinase A (PKA) and the exchange protein directly activated by cAMP (EPAC) dependent pathways in the regulation of NHE3 by adenosine 3',5'-cyclic monophosphate (cAMP). Opossum kidney cells and murine kidney slices were treated with cAMP analogs, which selectively activate either PKA or EPAC. Activation of either pathway resulted in an inhibition of NHE3 activity. The EPAC-induced effect was independent of PKA as indicated by the lack of activation of the kinase and the insensitivity to the PKA inhibitor H89. Both PKA and EPAC inhibited NHE3 activity without inducing changes in the expression of the transporter in BBM. Activation of PKA, but not of EPAC, led to an increase of NHE3 phosphorylation. In contrast, activation of PKA, but not of EPAC, inhibited renal type IIa Na(+)-coupled inorganic phosphate cotransporter (NaPi-IIa), another Na-dependent transporter expressed in proximal BBM. PKA, but not EPAC, induced the retrieval of NaPi-IIa from BBM. Our results suggest that EPAC activation may represent a previously unrecognized mechanism involved in the cAMP regulation of NHE3, whereas regulation of NaPi-IIa is mediated by PKA but not by EPAC.

Nateglinide uptake by a ceftibuten transporter in the rat kidney brush-border membrane

Nateglinide, a novel oral hypoglycemic agent, possesses a carbonyl group and a peptide-type bond in its structure. We previously reported that nateglinide transport occurs via a single system that may be identical to the ceftibuten/H(+) cotransport system by the rat small intestine. We speculated that the absorption system present on the intestinal epithelium may be similar to that found on the renal tubular epithelium. The aim of this study was to characterize the transporters on the apical side of the kidney that may contribute to the reabsorption of ceftibuten and nateglinide. The uptake of nateglinide by rat renal brush-border membranes is associated with an H(+)-coupled transport system. Ceftibuten competitively inhibited H(+)-dependent nateglinide uptake. In contrast, Gly-Sar, cephradine and cephalexin had no effect on nateglinide uptake. Nateglinide competitively inhibited H(+)-driven transporter-mediated ceftibuten uptake. We conclude that nateglinide transport occurs via a single system that is H(+)-dependent and may be identical to the ceftibuten/H(+) cotransport system.

Morphological, molecular, and functional changes in the chicken small intestine of the late-term embryo

The rapid development of the gastrointestinal tract posthatch has been described; however, little information exists concerning the development of the small intestine in the prehatch period. The present study examined the morphological, cellular, and molecular changes occurring in the small intestine toward the end of the incubation period by examining the expression of intestinal genes that code for brush border digestive enzymes and transporters, their biochemical activities, and the morphological changes in the mucosal layer. The results indicated that during the last 3 d of incubation the weight of the intestine, as a proportion of embryo weight, increased from approximately 1% on d 17 of embryonic age to 3.5% at hatch. At this time the villi could be divided into two main developmental stages, differing in their length and shape, with the larger villi often being pear-shaped and the smaller villi being narrower and having a rocket-like shape. However, on d 19 a further stage of villus development was observed. Activities of maltase, aminopeptidase, sodium-glucose transporter (SGLT)-1, and ATPase began to increase on d 19 and further increased on the day of hatch. The expression of mRNA for these brush-border membrane (BBM) enzymes and transporters was detected from d 15. Determining quantities relative to beta-actin indicated that expression of all parameters examined was low on d 15 and 17, increased 9- to 25-fold on d 19, and all decreased again on the day of hatch. Relative expression of mRNA of the different enzymes and transporters were correlated as were their activities (r = 0.75 to 0.96); however, expression was not correlated with enzymatic activities. The role of these parameters in the ontogeny of absorption is discussed. Thus, major changes in the expression and localization of the functional brush-border proteins prepare the framework for ingestion of carbohydrate- and protein-rich exogenous feed posthatch.

Cd-MT causes endocytosis of brush-border transporters in rat renal proximal tubules.

Nephrotoxicity in humans and experimental animals due to chronic exposure to cadmium (Cd) is manifested by defects in the reabsorptive and secretory functions of proximal tubules (PT). The main symptoms of Cd nephrotoxicity, including polyuria, phosphaturia, aminoaciduria, glucosuria, and proteinuria, suggest that various brush-border membrane (BBM) transporters are the main targets of Cd. Specific transporters may be either directly inhibited by Cd or lost from the BBM after Cd treatment, or both. We have recently proposed that Cd may impair the vesicle-dependent recycling of BBM transporters by inhibiting vacuolar H+-ATPase (V-ATPase) activity and endocytosis in PT cells (Herak-Kramberger CM, Sabolic I, and Brown D. Kidney Int 53: 1713-1726, 1998). The mechanism underlying the Cd effect was further explored in an in vivo model of experimental Cd nephrotoxicity induced by Cd-metallothionein (Cd-MT; 0.4 mg Cd/kg body mass; a single dose sc) in rats. The time-dependent redistribution of various BBM transporters was examined in this model by fluorescence and gold-labeling immunocytochemistry on tissue sections and by immunoblotting of isolated renal cortical BBM. In PT cells of Cd-MT-treated rats, we observed 1) shortening and loss of microvilli; 2) time-dependent loss of megalin, V-ATPase, aquaporin-1 (AQP1), and type 3 Na+/H+ exchanger (NHE3) from the BBM; 3) redistribution of these transporters into vesicles that were randomly scattered throughout the cell cytoplasm; and 4) redistribution of NHE3, but not megalin, into the basolateral plasma membrane. The internalization of BBM transporters was accompanied by fragmentation and loss of microtubules and by an increased abundance of alpha-tubulin monomers in PT cells. Transporter redistribution was detectable as early as 1 h after Cd-MT treatment and increased in magnitude over the next 12 h. We conclude that the early mechanism of Cd toxicity in PT cells may include a colchicine-like depolymerization of microtubules and impaired vesicle-dependent recycling of various BBM proteins. These processes may lead to a time-dependent loss of cell membrane components, resulting in reabsorptive and secretory defects that occur in Cd-induced nephrotoxicity.