Regulation of renal function by the gastrointestinal tract: potential role of gut-derived peptides and hormones.

The concept of a regulatory link between the gastrointestinal tract and kidneys is not new. The idea that dietary intake and composition can affect renal function is perhaps self-evident, but defining this relationship, especially in terms of sensors and effectors, is proving more difficult. That the gastrointestinal tract can exert some control over renal function was strengthened by the early observation that oral ingestion of a sodium chloride load has a greater natriuretic effect than when the same amount is given intravenously. This effect was subsequently shown to be independent of changes in aldosterone and atrial natriuretic peptide, although not necessarily angiotensin-II. However, the nature of any intestinal natriuretic peptide remains uncertain, despite suggestions that various gut-derived hormones, including guanylin and uroguanylin, may be involved. There is also an emerging concept of gastrointestinal taste and taste-like receptor mechanisms present throughout the gastrointestinal tract, which may govern the excretion of other key electrolytes, including potassium and phosphate. The evidence for gut sensors of nutrients such as proteins, amino acids, glucose, and acid is now becoming more established. Thus, we can anticipate the existence and eventual characterization of several gut ion sensors.

GLUT2 protein at the rat proximal tubule brush border membrane correlates with protein kinase C (PKC)-betal and plasma glucose concentration.

AIMS/HYPOTHESIS: GLUT2 is the main renal glucose transporter upregulated by hyperglycaemia, when it becomes detectable at the brush border membrane (BBM). Since glucose-induced protein kinase C (PKC) activation in the kidney is linked to diabetic nephropathy, we investigated the effect of glycaemic status on the protein levels of PKC isoforms alpha, betaI, betaII, delta and epsilon in the proximal tubule, as well as the relationship between them and changes in GLUT2 production at the BBM. METHODS: Plasma glucose concentrations were modulated in rats by treatment with nicotinamide 15 min prior to induction of diabetes with streptozotocin. Levels of GLUT2 protein and PKC isoforms in BBM were measured by western blotting. Additionally, the role of calcium signalling and PKC activation on facilitative glucose transport was examined by measuring glucose uptake in BBM vesicles prepared from proximal tubules that had been incubated either with thapsigargin, which increases cytosolic calcium, or with the PKC activator phorbol 12-myristate,13-acetate (PMA). RESULTS: Thapsigargin and PMA enhanced GLUT-mediated glucose uptake, but had no effect on sodium-dependent glucose transport. Diabetes significantly increased the protein levels of GLUT2 and PKC-betaI at the BBM. Levels of GLUT2 and PKC-betaI correlated positively with plasma glucose concentration. Diabetes had no effect on BBM levels of alpha, betaII, delta or epsilon isoforms of PKC. CONCLUSIONS/INTERPRETATION: Enhanced GLUT2-mediated glucose transport across the proximal tubule BBM during diabetic hyperglycaemia is closely associated with increased PKC-betaI. Thus, altered levels of GLUT2 and PKC-betaI proteins in the BBM may be important factors in the pathogenic processes underlying diabetic renal injury.

Intestinal phosphate absorption in a model of chronic renal failure.

Hyperphosphatemia is an important consequence of chronic renal failure (CRF). Lowering of the plasma phosphate concentration is believed to be critical in the management of patients with CRF, especially those on dialysis. Reports of the effect of CRF on the intestinal handling of phosphate in vitro have been conflicting; but what happens in vivo has not been studied. What effect a reduction in the dietary phosphate intake has on intestinal phosphate absorption in CRF in vivo is unclear. In this study, we have used the in situ intestine loop technique to determine intestinal phosphate absorption in the 5/6-nephrectomy rat model of CRF under conditions of normal and restricted dietary phosphate intake. In this model of renal disease, we found that there is no significant change in the phosphate absorption in either the duodenum or jejunum regardless of the dietary phosphate intake. There was also no change in the expression of the messenger RNA of the major intestinal phosphate carrier the sodium-dependent-IIb transporter. Furthermore, we found no change in the intestinal villus length or in the location of phosphate uptake along the villus. Our results indicate that in CRF, unlike the kidney, there is no reduction in phosphate transport across the small intestine. This makes intestinal phosphate absorption a potential target in the prevention and treatment of hyperphosphatemia.

Non-haem iron transport in the rat proximal colon.

BACKGROUND: Only 10% of dietary iron is absorbed in the duodenum which implies that 90% (approximately 9 mg day(-1)) reaches the lower small intestine and colon. Therefore the purpose of this study was to assess the iron transport capacity of the rat proximal colon and to determine whether iron absorption is regulated by changes in dietary iron content. MATERIALS AND METHODS: Rats were fed for 14 days on either iron adequate (44 mg Fe kg(-1) diet) or iron-deficient (< 0.5 mg Fe kg(-1) diet) diets. The 59Fe transport across the colonic epithelium and its subsequent appearance in the blood were measured in vivo. In separate studies the colon was excised and used to measure divalent metal transporter expression. RESULTS: Divalent metal transporter (DMT1) was expressed at the apical membrane of the surface epithelium in rat proximal colon. In animals fed an iron-deficient diet, DMT1 mRNA and protein expression were increased. This was accompanied by a significant increase in tissue 59Fe uptake. CONCLUSIONS: The proximal colon can absorb non-haem iron from the intestinal lumen. The purpose of this mechanism remains to be elucidated.

Increased duodenal iron uptake and transfer in a rat model of chronic hypoxia is accompanied by reduced hepcidin expression.

BACKGROUND: Despite the requirement for increased iron delivery for erythropoiesis during hypoxia, there is very little information on how duodenal iron uptake and its transfer to the blood adapts to this condition. AIMS: To assess the effects of 30 days of chronic hypoxia in rats on luminal iron uptake and transfer of the metal to blood, together with gene expression of hepcidin, a proposed negative regulator of iron transport. METHODS: 59-Fe uptake by isolated duodenum and its transfer to blood by in vivo duodenal segments was measured after exposure of rats to room air or 10% oxygen for four weeks. Liver hepcidin expression was measured by real time reverse transcription-polymerase chain reaction. The effects of hypoxia on hepcidin gene expression by HepG2 cells was also determined. RESULTS: Hypoxia did not affect villus length but enhanced (+192.6%) luminal iron uptake by increasing the rate of uptake by all enterocytes, particularly those on the upper villus. Hypoxia promoted iron transfer to the blood but reduced mucosal iron accumulation in vivo by 66.7%. Hypoxia reduced expression of hepcidin mRNA in both rat liver and HepG2 cells. CONCLUSIONS: Prolonged hypoxia enhances iron transport from duodenal lumen to blood but the process is unable to fully meet the iron requirement for increased erythropoiesis. Reduced secretion of hepcidin may be pivotal to the changes in iron absorption. The processes responsible for suppression of hepcidin expression are unknown but are likely to involve a direct effect of hypoxia on hepatocytes.

Detection of glucagon receptor mRNA in the rat proximal tubule: potential role for glucagon in the control of renal glucose transport.

Glucagon is known to affect glomerular filtration rate and renal tubular solute and fluid transport, although it is only thought to act directly on the thick ascending limb (TAL) and collecting duct (CD). Indeed, previous studies have detected glucagon-sensitive adenylate cyclase exclusively in these nephron segments, suggesting the presence of glucagon receptors. In the present study, we have demonstrated for the first time that glucagon receptor mRNA is expressed in the rat proximal tubule, as well as in the TAL and CD. By autoradiography, we have also shown that specific binding of glucagon occurs in both the renal cortex and medulla. In addition, using proximal tubule brush-border membrane (BBM) vesicles for studies of glucose transport, we have established that glucagon stimulates glucose uptake via a facilitative GLUT-mediated transport process (by 58%; P < 0.005), whereas cAMP stimulates only the sodium glucose-linked transporter ('SGLT')-mediated glucose uptake (by 53%; P < 0.05). Taken together, these findings suggest that glucagon could have a role in controlling proximal tubular transport function, including glucose reabsorption, but unlike in the TAL and CD, the proximal tubule glucagon receptor might not be coupled primarily to adenylate cyclase.

Methods for assessing intestinal absorptive function in relation to enteral nutrition.

The success of nasoenteral nutrition support can be limited by intestinal impairment. In particular, reduced absorptive area, mucosal atrophy and abnormal motility may reduce absorption of macronutrients and micronutrients, and diarrhoea remains a commonly encountered complication. We review how basic physiological techniques can be used to investigate such pathophysiology. Lumenal nutrients control mucosal growth, expression of mucosal transporters and regional gut motility. Cell biology techniques now complement classical intestinal perfusion methods in determining the 'safety factor' of excess absorptive capacity. The controversial role of the sodium-glucose linked transporter in dietary glucose assimilation is described in terms of its control, its true function and its role in uptake of other solutes. Techniques that involve brush-border membrane vesicles, Caco-2 cells, mucosal immunohistochemistry and gene expression probes are described. Together, these techniques describe a picture of an organ with remarkable ability to maintain digestive and absorptive function in response to a wide variety of nutritional intakes, often in the face of inflammatory illness.

Epicatechin is the primary bioavailable form of the procyanidin dimers B2 and B5 after transfer across the small intestine.

Perfusion of isolated small intestine with the procyanidin dimers B2 and B5 extracted from cocoa indicated that both forms of dimer are transferred to the serosal side of enterocytes but only to a very small extent (<1% of the total transferred flavanol-like compounds). However, perfusion of dimer mainly resulted in large amounts of unmetabolised/unconjugated epicatechin monomer being detected on the serosal side (95.8%). The cleavage of dimer during transfer seemed to be energy-dependent, requiring an intact cell system, as incubation with jejunal homogenates failed to yield epicatechin. Low levels methylated dimer were also detected (3.2%), but no conjugates and metabolites of epicatechin indicating that metabolism of monomer and dimer is limited during dimer cleavage/translocation. The methylation of dimer may be by catechol-O-methyltransferase, however, at high concentrations of dimer COMT activity is reduced leading to an inhibition of both monomer and dimer O-methylation.

Epicatechin and catechin are O-methylated and glucuronidated in the small intestine.

There is considerable interest in the bioavailability of polyphenols and their bioactivity in vivo. We have studied the absorption and metabolism of catechin and epicatechin in the small intestine and the comparative transfer across the jejunum and ileum. Perfusion of isolated jejunum with the flavanols resulted in glucuronidation ( approximately 45%), O-methylation: 3'-O-Methyl- and 4'-O-methyl- ( approximately 30%), and O-methyl-glucuronidation ( approximately 20% of total flavanols identified) during transfer across the enterocytes to the serosal side. This demonstrates the activity of catechol-O-methyl transferases in the metabolism of flavanols and suggests that these metabolites and conjugates are likely to enter the portal vein. In contrast, in the case of the ileum, the majority of the flavanols appeared on the serosal side unmetabolised and the total percentage of flavanols transferred was higher than that in the jejunum ( approximately fivefold).

Resveratrol is absorbed in the small intestine as resveratrol glucuronide.

We have studied the absorption and metabolism of resveratrol in the jejunum in an isolated rat small intestine model. Only small amounts of resveratrol were absorbed across the enterocytes of the jejunum and ileum unmetabolised. The major compound detected on the serosal side was the glucuronide conjugate of resveratrol (96.5% +/- 4.6 of the amount absorbed) indicating the susceptibility of resveratrol to glucuronidation during transfer across the rat jejunum. The presence of the glucuronide was confirmed using HPLC-PDA and nanoES-MS/MS techniques. These findings suggest that resveratrol is most likely to be in the form of a glucuronide conjugate after crossing the small intestine and entering the blood circulation. This will have important implications for the biological functions of resveratrol in vivo.

Structural and cellular adaptation of duodenal iron uptake in rats maintained on an iron-deficient diet.

Iron deficiency induced in rats maintained on a commercial diet with a low iron content has been used to investigate adaptive mechanisms that enhance duodenal iron uptake. These adaptive changes have been divided into those that result from changes in villus surface area (structural adaptation) and those that reflect changes in the way individual enterocytes express iron transport function (cellular adaptation). Cellular adaptation was assessed by carrying out microdensitometry of autoradiographs prepared from duodenal tissue previously incubated for 5 min in 200 micromol/l 59Fe2+-ascorbate. Structural adaptation was studied by performing image analysis of microdissected and sectioned villi. Cellular adaptation involved increased iron uptake by enterocytes present in the lower villus. Thus iron deficiency resulted in a threefold enhanced expression of uptake in the lower 100 microm villus (3.9+/-2.4 versus 12.6+/-1.5 arbitrary units, P<0.001). Maximal uptake was reached in the upper region of both control and iron-deficient villi, but iron deficiency had no effect on cellular uptake at this part of the villus. Structural adaptation involved the lengthening (+16%, P<0.05) and broadening (+14%) of villi in the duodenum of iron-deficient rats. The resultant expansion in villus area caused a further increase in uptake that was mostly expressed in the upper villus. Maximal uptake corrected for structure occurred in the middle third of villi from control and iron-deficient rats. Cellular plus structural adaptation produced a twofold increase in iron uptake. More than half of this effect was caused by changes in villus structure. [3H]Thymidine labelling experiments revealed a slightly earlier expression of enterocyte iron uptake in iron deficiency.

The small intestine can both absorb and glucuronidate luminal flavonoids.

We have studied the perfusion of the jejunum and ileum in an isolated rat intestine model with flavonoids and hydroxycinnamates and the influence of glycosylation on the subsequent metabolism. Flavone and flavonol glucosides and their corresponding aglycones are glucuronidated during transfer across the rat jejunum and ileum and this glucuronidation occurs without the need for gut microflora. Furthermore, this suggests the presence of glycosidases as well as UDP-glucuronyl transferase in the jejunum. In contrast, quercetin-3-glucoside and rutin are mainly absorbed unmetabolised. The results suggest that the more highly reducing phenolics are absorbed predominantly as glucuronides (96.5%+/-4.6) of the amount absorbed, whereas monophenolic hydroxycinnamates and monophenolic B-ring flavonoids are less predisposed to glucuronidation and higher levels of aglycone (88.1%+/-10.1) are detected on absorption through both the jejunum and ileum.

Acute and chronic exposure of rat intestinal mucosa to dextran promotes SGLTI-mediated glucose transport.

BACKGROUND: The intestinal handling of dextran, an alpha-1,6-linked glucose polymer, is poor compared with starch, and some ingested dextran might therefore reach the lower small intestine. As luminal sugar up-regulates SGLT1 (sodium-dependent glucose transporter) locally, we report the effects of a dextran-enriched diet on jejunal and ileal brush border membrane (BBM) glucose uptake. METHODS: Rats were maintained on a diet containing 65% maltodextrin or 32.5% maltodextrin + 32.5% dextran (10 kD or 40 kD) for 8-10 days, and the kinetics of phlorizin-sensitive [3H]-glucose uptake by purified BBM vesicles was determined. RESULTS: Ingestion of 40-kD but not 10-kD dextran increased Vmax for jejunal and ileal glucose uptake (+64.3% and +61.8% respectively, both P < 0.02). The transport response to 40-kD dextran was in keeping with lower levels of expired H2 at the end of the feeding period. High-performance liquid chromatography (HPLC) analysis of luminal contents indicated extensive hydrolysis of ingested dextran. Finally, 3-h jejunal exposure to 40-kD dextran in vivo increased the Vmax for glucose uptake by jejunal BBM. CONCLUSION: It is likely that increased SGLT1-mediated glucose uptake after short or longer term mucosal exposure to dextran results from luminal dextran per se or a hydrolysis product. The clinical implications of this up-regulation are discussed.

Early diabetes-induced changes in rat jejunal glucose transport and the response to insulin.

The effects of 1 day of streptozotocin-induced diabetes in rats on glucose transport across the brush border membrane (BBM) and basolateral membrane (BLM) prepared from jejunal enterocytes has been studied. The effects on glucose transport of treatment of diabetic animals with insulin to reduce to normal the elevated blood glucose levels has also been assessed. The maximum capacity (Vmax) for SGLT1-mediated glucose uptake by BBM vesicles was unaffected by diabetes or insulin treatment of diabetic rats. In contrast, Vmax for BLM glucose uptake was increased by 206% in diabetes, a response that could not be reversed by treatment with insulin. Western blotting of BBM for SGLT1 protein revealed a single band with a molecular weight of 73 kDa and the intensity of this band was unaffected by diabetes. However, an increased level of GLUT2 was noted in diabetic BLM and this was not a consequence of changes in glycaemic or insulin status. Diabetes hyperpolarised the BBM, implying an increased driving force for Na(+)-sugar co-transport but insulin treatment only partially reversed this enhanced potential difference. Benzamil (2 microns), an epithelial Na+ channel blocker, hyperpolarised the BBM of control but not diabetic enterocytes, implying that a reduced Na+ permeability was responsible for the diabetic hyperpolarisation. It was concluded that in early diabetes, before the onset of hyperphagia, a greater driving force for Na(+)-dependent BBM sugar transport together with increased GLUT2 activity at the BLM promotes sugar movement across the enterocyte. Possible triggers for the transport responses are discussed.

Mechanisms involved in increased iron uptake across rat duodenal brush-border membrane during hypoxia.

1. Chronic hypoxia enhances intestinal iron transport but the cellular processes involved are poorly understood. In order to assess the effects of 3 days of hypoxia on iron uptake across the duodenal brush-border membrane, we have measured the membrane potential difference (Vm) of villus-attached enterocytes by direct microelectrode impalement and have used semi-quantitative autoradiography to study changes in expression of iron uptake during enterocyte maturation. 2. Hypoxia increased duodenal Vm (-57.7 vs. -49.3 mV, P < 0.001). Ion substitution experiments revealed that hyperpolarization was due, at least in part, to a reduction in brush-border Na+ permeability. 3. Autoradiography revealed that hypoxia increased by 6-fold the rate of iron accumulation during enterocyte transit along the lower villus and enhanced by 3-fold the maximal accumulation of iron. Depolarization of the brush border, using a high-K(+)-containing buffer, caused a proportionally greater reduction in iron uptake in control compared with hypoxic tissue suggesting that the raised iron uptake is only partly driven by brush-border hyperpolarization. 4. We conclude that hypoxia increases the expression of iron transport in duodenal brush-border membrane and an enhanced electrical driving force may be involved in this response.

Streptozotocin diabetes and the expression of GLUT1 at the brush border and basolateral membranes of intestinal enterocytes.

Changes in membrane expression of sodium-dependent glucose transporter (SGLT1) and glucose transporter isoform (GLUT2) protein have been implicated in the increased intestinal glucose transport in streptozotocin-diabetes. The possible involvement of GLUT1 in the transport response, however, has not previously been studied. Using confocal microscopy on tissue sections and Western blotting of purified brush border membrane (BBM) and basolateral membrane (BLM), we have examined enterocyte expression of GLUT1 in untreated and in 1 and 21 day streptozotocin diabetic rats. In control enterocytes, GLUT1 was absent at the BBM and detected at low levels at the BLM. Diabetes resulted in a 4- to 5-fold increased expression of GLUT1 at the BLM and the protein could also be readily detected at the BBM. Insulin treatment of diabetic rats increased GLUT1 level at the BBM but was without effect on expression of the protein at the BLM.

Rapid enhancement of brush border glucose uptake after exposure of rat jejunal mucosa to glucose.

BACKGROUND: Increased jejunal glucose transport after ingestion of carbohydrate rich diets may reflect higher concentrations of lumenal glucose. Normal processing of carbohydrate causes wide fluctuations in glucose concentration in the jejunal lumen and this raises the question of whether the high lumenal concentrations seen at peak digestion affect glucose uptake. AIMS: To study the effects of 30 minute exposure of rat jejunal mucosa to glucose on sodium-glucose transporter (SGLT1) mediated glucose transport across the brush border membrane. METHODS: Jejunal mucosa was exposed in vitro or in vivo to 25 mM glucose or 25 mM mannitol for 30 minutes. In addition, isolated villus enterocytes were incubated with mannitol or glucose for the same time. Brush border membrane vesicles were isolated from these preparations and phlorizin sensitive 3H-D-glucose accumulation was measured. RESULTS: Lumenal glucose in vivo significantly enhanced SGLT1 mediated glucose uptake by 49.2-57.2%. For jejunal loops in vitro, the increase was 32.0-85.2%. Kinetic analysis disclosed a 50% greater Vmax for glucose uptake in each preparation. The facilitated and passive components of uptake were, however, unaffected by prior exposure to glucose. Incubation of villus enterocytes with 25 mM glucose did not influence glucose uptake by brush border membranes. Finally, exposure of intact mucosa to 20 mM galactose, a nonmetabolised sugar also transported by SGLT1, did not alter glucose transport. CONCLUSIONS: Lumenal glucose promotes glucose transport by brush border membrane within 30 minutes. An intact mucosa is necessary for upregulation and evidence suggests that the response is mediated by locally acting mechanisms.

Cellular mechanisms underlying the increased duodenal iron absorption in rats in response to phenylhydrazine-induced haemolytic anaemia.

Haemolytic anaemia induced by phenylhydrazine (PZ) promotes iron absorption across rat small intestine. This present study investigates the role of the brush border potential difference (Vm) and mucosal reducing activity in the response. In addition, quantitative autoradiography was used to assess PZ-induced changes in the villus localization of brush border iron uptake. Iron transfer from duodenum to blood was increased significantly 5 days after treatment with PZ. Autoradiography showed that most brush border iron uptake occurred at the upper villus region and the maximal rate was increased fourfold by PZ. Duodenal villus length was increased in PZ-treated rats. PZ treatment did not influence mucosal reducing activity but Vm, measured using duodenal sheets, increased from -50 to -57 mV (P < 0.001) and this was due to a reduced brush border sodium permeability. Thus, an expanded absorptive surface and an enhanced electrical driving force for iron uptake across the duodenal brush border are important adaptations for increased iron absorption in PZ-induced haemolytic anaemia.