Net Secretion Research Articles

Metabolomic and Lipidomic Characterization of Oxalobacter formigenes to Define Intestinal Oxalate Secretion

Kidney stone disease afflicts 1 in 11 people in the U.S. and burdens the economy with an annual treatment cost of over $10 billion. Nearly 80% of kidney stones are composed of calcium oxalate (CaOx) and form when humans are exposed to oxalate through the diet and endogenous glyoxylate metabolism in the liver. Recently, significant clinical interest has turned to the intestinal microbiome, specifically a class of “oxalotrophic” bacteria that degrade dietary oxalate in the intestine. These bacteria reduce the amount of oxalate absorbed by the body, lowering circulating and urinary oxalate, both which directly influence CaOx stone formation. One species in particular, Oxalobacter formigenes (Oxf), solely utilizes oxalate as an energy source and has the unique ability among oxalotrophs to transport endogenous oxalate from the bloodstream into the intestine to increase oxalate availability, allowing it to feed on both metabolic and dietary oxalate. The potential for Oxf as a probiotic therapy for kidney stone disease is currently being evaluated by numerous institutions. Compelling evidence demonstrates that Oxf produces some form of secreted bioactive compound to regulate oxalate transport proteins in the intestinal wall to bring oxalate across the epithelium. This compound, if identified, could potentially serve as a future therapy for kidney stone formation. This study seeks to identify this secreted factor through an Ultra High Performance Liquid Chromatography‐Mass Spectrometry‐based characterization and comparison of the metabolome and lipidome of Oxf with other oxalotrophs that cannot initiate oxalate secretion, the rationale being that the bioactive compound(s) responsible for this function should be uniquely expressed by Oxf. We also present the first characterization of the Oxf metabolome and lipidome by comparing the human strain HC1 and the wild rat strain OxWR, both which have been shown to induce net intestinal oxalate secretion across native intestinal epithelial tissue. It is essential to elucidate the molecular nature of the Oxf‐host relationship to bring this bacterium and its metabolic products to the clinic as a novel approach to kidney stone disease.Support or Funding InformationThis work was funded by the National Institutes of Health grant 2R01DK088892‐05A1.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Gout Causing ABCG2 Mutation Results in Intestinal Net Urate Transport Defect, Hyperuricemia, & Altered Metabolic Phenotype

Hyperuricemia causes gout and is implicated in metabolic syndrome, impaired glucose tolerance/type 2 diabetes, and hypertension. Individuals carrying a common loss of function variant in the dominant secretory transporter, Q141K ABCG2, have significantly increased serum urate and gout risk yet their renal fractional excretion of urate (FEUA) is largely unchanged. However, the role in ABCG2 and other urate transporters in extra‐renal secretion remains mostly unexplored. Here we sought to identify the role of ABCG2 in trans‐epithelial urate transport in the intestines and contribution of intestinal ABCG2 dysfunction to hyperuricemia and urate related phenotypes. First, we established a genetic mouse model of the human ABCG2 Q141K variant (Q140K in mouse) using CRISPR Cas9 gene editing techniques on a C57BL6 mouse background. As compared to control mice, mice possessing the Q140K Abcg2 allele were profoundly hyperuricemic, yet exhibited only modest changes in renal excretion of urate, similar to what has been reported previously in humans. To explore mechanisms, we first mapped prominent urate transporters ABCG2 and SLC2A9 along the mouse intestine and found the highest levels in the jejunum and ileum. Localization of the wild type ABCG2 protein was exclusive to the brush border (apical membrane) of the villus cells, seemingly optimized to facilitate urate secretion(JUAsm). Interestingly, we also found evidence that SLC2A9 localized predominantly to the basolateral enterocyte membrane. In Ussing chamber experiments, we functionally confirmed basolateral localization of the electrogenic SLC2A9 transporter by observing basolateral but not apical addition of 0.5 mM urate significantly stimulated Isc (24 ± 3μA/cm2) in comparison with control (10± 2.2 μA/cm2). Further, treatment of the serosal membrane with SLC2A9 inhibitors tranilast and pCMBS partially inhibited JUAsm flux of urate in an intestinal loop model. In contrast to the WT controls, Q140K mice demonstrated a significant decrease of apical ABCG2 protein abundance, with commiserate, significant loss of unidirectional urate transport (JUAsm) in jejunal loops (Q140K, 24 ± 3.38 μM/cm2/min; WT, 46 ± 1.50 μM/cm2/min), resulting in conversion of net urate secretion (−18 ± 4 μM/cm2/min) to net urate absorption (+5.99 ± 2.2 μM/cm2/min). Interestingly, these significant alterations in intestinal urate secretion in the Q140K mice correlated with evidence of altered serum insulin and non‐fasting glucose levels, not unlike what has been previously reported in mice lacking intestinal SLC2A9 strongly suggesting the establishment of SLC2A9 and ABCG2 as the intestinal urate excretion pathway.CONCLUSIONSOur findings demonstrate the pathophysiology of one of the most common hyperuricemia and gout risk variants, Q141K ABCG2, primarily affects the urate secretion in the gut, causing hyperuricemia and alterations in insulin and glucose levels with important implications for human disease.Support or Funding InformationNIDDK RO1DK114091AHA14SDG18060004This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolomic and lipidomic characterization of Oxalobacter formigenes strains HC1 and OxWR by UHPLC-HRMS.

Diseases of oxalate, such as nephrolithiasis and primary hyperoxaluria, affect a significant portion of the US population and have limited treatment options. Oxalobacter formigenes, an obligate oxalotrophic bacterium in the mammalian intestine, has generated great interest as a potential probiotic or therapeutic treatment for oxalate-related conditions due to its ability to degrade both exogenous (dietary) and endogenous (metabolic) oxalate, lowering the risk of hyperoxaluria/hyperoxalemia. Although all oxalotrophs degrade dietary oxalate, Oxalobacter formigenes is the only species shown to initiate intestinal oxalate secretion to draw upon endogenous, circulating oxalate for consumption. Evidence suggests that Oxalobacter regulates oxalate transport proteins in the intestinal epithelium using an unidentified secreted bioactive compound, but the mechanism of this function remains elusive. It is essential to gain an understanding of the biochemical relationship between Oxalobacter and the host intestinal epithelium for this microbe to progress as a potential remedy for oxalate diseases. This investigation includes the first profiling of the metabolome and lipidome of Oxalobacter formigenes, specifically the human strain HC1 and rat strain OxWR, the only two strains shown thus far to initiate net intestinal oxalate secretion across native gut epithelia. This study was performed using untargeted and targeted metabolomics and lipidomics methodologies utilizing ultra-high-performance liquid chromatography-mass spectrometry. We report our findings that the metabolic profiles of these strains, although largely conserved, show significant differences in their expression of many compounds. Several strain-specific features were also detected. Discussed are trends in the whole metabolic profile as well as in individual features, both identified and unidentified. Graphical abstract ᅟ.

Differential glycogen utilization in shark acid- and base-regulatory gill cells.

Na+/K+-ATPase (NKA)- and vacuolar H+-ATPase (VHA)-rich cells in shark gills secrete excess acid and base, respectively, to seawater to maintain blood acid-base homeostasis. Both cell types are rich in mitochondria, indicating high ATP demand; however, their metabolic fuel is unknown. Here, we report that NKA- and VHA-rich cells have large glycogen stores. Glycogen abundance in NKA-rich cells was lower in starved sharks compared with 24 h post-fed sharks, reflecting higher energy demand for acid secretion during normal activity and glycogen replenishment during the post-feeding period. Conversely, glycogen abundance in VHA-rich cells was high in starved sharks and it became depleted post-feeding. Furthermore, inactive cells with cytoplasmic VHA had large glycogen stores and active cells with basolateral VHA had depleted glycogen stores. These results indicate that glycogen is a main energy source in both NKA- and VHA-rich cells, and point to differential energy use associated with net acid and net base secretion, respectively.

Characterization of renal NaCl and oxalate transport in Slc26a6-/- mice.

The apical membrane Cl-/oxalate exchanger SLC26A6 has been demonstrated to play a role in proximal tubule NaCl transport based on studies in microperfused tubules. The present study is directed at characterizing the role of SLC26A6 in NaCl homeostasis in vivo under physiological conditions. Free-flow micropuncture studies revealed that volume and Cl- absorption were similar in surface proximal tubules of wild-type and Slc26a6-/- mice. Moreover, the increments in urine flow rate and sodium excretion following thiazide and furosemide infusion were identical in wild-type and Slc26a6-/- mice, indicating no difference in NaCl delivery out of the proximal tubule. The absence of an effect of deletion of SLC26A6 on NaCl homeostasis was further supported by the absence of lower blood pressure in Slc26a6-/- compared with wild-type mice on normal or low-salt diets. Moreover, raising plasma and urine oxalate by feeding mice a diet enriched in soluble oxalate did not affect mean blood pressure. In contrast to the lack of effect of SLC26A6 deletion on NaCl homeostasis, fractional excretion of oxalate was reduced from 1.6 in wild-type mice to 0.7 in Slc26a6-/- mice. We conclude that, although SLC26A6 is dispensable for renal NaCl homeostasis, it is required for net renal secretion of oxalate.

Using a semi-mechanistic model to identify the main sources of variability of metformin pharmacokinetics.

Metformin pharmacokinetics (PK) is highly variable, and researchers have for years tried to shed light on determinants of inter-individual (IIV) and inter-occasion variability (IOV) of metformin PK. We set out to identify the main sources of PK variability using a semi-mechanistic model. We assessed the influence of subject characteristics, including seven genetic variants. Data from three studies of healthy individuals with PK measurements of plasma and urine after single dose or at steady-state were used in this study. In total, 87 subjects were included (16 crossover subjects). Single nucleotide polymorphisms in ATM, OCT1, OCT2, MATE1 and MATE2-K were investigated as dominant, recessive or additive. A three-compartment model with transit absorption and renal elimination with a proportional error was fitted to the data using NONMEM 7.3. Oral parameters were separated from disposition parameters as dose-dependent absolute bioavailability was determined with support from urine data. Clearance was expressed as net renal secretion and filtration, assuming full fraction unbound and fraction excreted. Mean transit time and peripheral volume of distribution were identified as the main sources of variability according to estimates, with 94% IOV and 95% IIV, respectively. Clearance contributed only with 16% IIV. Glomerular filtration rate and body-weight were the only covariates found to affect metformin net secretion, reducing IIV to 14%. None of the genetic variants were found to affect metformin PK. Based on our analysis, finding covariates explaining absorption of metformin is much more valuable in understanding variability and avoiding toxicity than elimination.

Uteroplacental secretion of progesterone and estradiol-17β in an ovine model of intrauterine growth restriction

Using a mid to late gestation model of intrauterine growth restriction, uteroplacental secretion of progesterone and estradiol-17β were examined. From day 50 to 130 of gestation, 31 ewe lambs were allocated to receive 100% (ADQ) or 60% (RES) of nutrient requirements. At day 130, umbilical and uterine artery blood flows were determined and blood samples were collected from maternal saphenous artery, gravid uterine vein, umbilical vein, and umbilical artery. Uteroplacental secretion of progesterone was increased in RES compared to ADQ fed dams. There was a net secretion and net metabolism of estradiol-17β in RES, and ADQ fed dams, respectively. In relation to steroid synthesis, cotyledonary abundance of steroidogenic acute regulatory protein was greater in RES compared with ADQ fed dams, while abundance of aromatase was not different between dietary treatments. Caruncular aldo-keto reductase 1C abundance was less in RES compared to ADQ fed dams. The increase in progesterone secretion, therefore, is due in part to an increase in synthesis and a decrease in placental catabolism. Caruncular cytochrome P450 3A, which catalyzes the conversion of estrogens to catechol-estrogens, was in lesser abundance in RES compared to ADQ fed dams. Opposite responses in estradiol-17β uteroplacental secretion compared with metabolism may be mediated through placental estrogen metabolism via cytochrome P450 enzymes.

Reduced active transcellular intestinal oxalate secretion contributes to the pathogenesis of obesity-associated hyperoxaluria

Most kidney stones are composed of calcium oxalate, and minor changes in urine oxalate affect the stone risk. Obesity is a risk factor for kidney stones and a positive correlation of unknown etiology between increased body size, and elevated urinary oxalate excretion has been reported. Here, we used obese ob/ob (ob) mice to elucidate the pathogenesis of obesity-associated hyperoxaluria. These ob mice have significant hyperoxaluria (3.3-fold) compared with control mice, which is not due to overeating as shown by pair-feeding studies. Dietary oxalate removal greatly ameliorated this hyperoxaluria, confirming that it is largely enteric in origin. Transporter SLC26A6 (A6) plays an essential role in active transcellular intestinal oxalate secretion, and ob mice have significantly reduced jejunal A6 mRNA (- 80%) and total protein (- 62%) expression. While net oxalate secretion was observed in control jejunal tissues mounted in Ussing chambers, net absorption was seen in ob tissues, due to significantly reduced secretion. We hypothesized that the obesity-associated increase in intestinal and systemic inflammation, as reflected by elevated proinflammatory cytokines, suppresses A6-mediated intestinal oxalate secretion and contributes to obesity-associated hyperoxaluria. Indeed, proinflammatory cytokines (elevated in ob mice) significantly decreased intestinal oxalate transport invitro by reducing A6 mRNA and total protein expression. Proinflammatory cytokines also significantly reduced active mouse jejunal oxalate secretion, converting oxalate transport from net secretion in vehicle-treated tissues to net absorption in proinflammatory cytokines-treated tissues. Thus, reduced active intestinal oxalate secretion, likely secondary to local and systemic inflammation, contributes to the pathogenesis of obesity-associated hyperoxaluria. Hence, proinflammatory cytokines represent potential therapeutic targets.

Short-chain fatty acids augment rat duodenal mucosal barrier function.

What is the central question of this study? Small intestinal epithelium is exposed to high concentrations of short-chain fatty acids (SCFAs), but their role in regulating intestinal mucosal barrier function and motility is not fully understood. What is the main finding and its importance? By perfusing the duodenal segment in anaesthetized rats, we show that acetate and propionate significantly decrease mucosal paracellular permeability and transepithelial net fluid flux and increase mucosal bicarbonate secretion. Likewise, SCFAs administered i.v. decrease mucosal permeability but decrease bicarbonate secretion. Altered luminal chemosensing or aberrant signalling in response to SCFAs might contribute to symptoms observed in patients with suppressed mucosal barrier function. Short-chain fatty acids (SCFAs) are produced by bacterial fermentation in the large intestine, particularly from diets containing fibres and carbohydrates. The small intestinal epithelium is exposed to SCFAs derived mainly from oral bacteria or food supplementation. Although luminal nutrients are important in regulation of intestinal functions, the role of SCFAs in regulation of small intestinal mucosal barrier function and motility has not been fully described. The aim of the present study was to elucidate the effects of acetate and propionate on duodenal mucosal barrier function and motility. Rats were anaesthetized with thiobarbiturate, and a 30mm segment of proximal duodenum with an intact blood supply was perfused. The effects on duodenal bicarbonate secretion, blood-to-lumen clearance of 51 Cr-EDTA, motility and transepithelial net fluid flux were investigated. Perfusion of the duodenum with acetate or propionate significantly decreased mucosal paracellular permeability and transepithelial net fluid flux and significantly increased bicarbonate secretion. Acetate or propionate administered as an i.v. infusion decreased the mucosal paracellular permeability, but significantly decreased bicarbonate secretion. Luminal SCFAs changed the duodenal motility pattern from migrating motor complexes to fed patterns. Systemic administration of glucagon-like peptide-2 induced increases in both bicarbonate secretion and net fluid absorption, but did not change motility. Glucagon-like peptide-2 infusion during luminal perfusion of SCFAs significantly reduced the motility. In conclusion, SCFAs decreased duodenal paracellular permeability and net fluid flux. Short-chain fatty acids induced opposite effects on bicarbonate secretion after luminal and i.v. administration. Presence of SCFAs in the lumen induces fed motility patterns. Altered luminal chemosensing and aberrant signalling in response to SCFAs might contribute to symptoms observed in patients with suppressed barrier function.

Glucose enhances rotavirus enterotoxin-induced intestinal chloride secretion.

Rotavirus causes severe diarrhea in small children and is typically treated using glucose-containing oral rehydration solutions; however, glucose may have a detrimental impact on these patients, because it increases chloride secretion and presumably water loss. The rotavirus enterotoxin nonstructural protein 4 (NSP4) directly inhibits glucose-mediated sodium absorption. We examined the effects of NSP4 and glucose on sodium and chloride transport in mouse small intestines and Caco-2 cells. Mouse small intestines and Caco-2 cells were incubated with NSP4114-135 in the presence/absence of glucose. Absorption and secretion of sodium and chloride, fluid movement, peak amplitude of intracellular calcium fluorescence, and expression of Ano1 and sodium-glucose cotransporter 1 were assessed. NHE3 activity increased, and chloride secretory activity decreased with age. Net chloride secretion increased, and net sodium absorption decreased in the intestines of 3-week-old mice compared to 8-week-old mice with NSP4. Glucose increased NSP4-stimulated chloride secretion. Glucose increased NSP4-stimulated increase in short-circuit current measurements (I sc) and net chloride secretion. Ano1 cells with siRNA knockdown showed a significant difference in I sc in the presence of NSP4 and glucose without a significant difference in peak calcium fluorescence intracellular when compared to non-silencing (N.S.) cells. The failure of glucose to stimulate significant sodium absorption was likely due to the inhibition of sodium-hydrogen exchange and sodium-glucose cotransport by NSP4. Since glucose enhances intestinal chloride secretion and fails to increase sodium absorption in the presence of NSP4, glucose-based oral rehydration solutions may not be ideal for the management of rotaviral diarrhea.

The Effect of Uremic Solutes on the Organic Cation Transporter 2

Chronic kidney disease (CKD) is characterized by the accumulation of uremic solutes; however, little is known about how these solutes affect drug absorption and disposition. The goal of this study is to evaluate the effect of uremic solutes on the organic cation transporter, OCT2, which plays a key role in the renal secretion of many basic drugs. As a second goal, we reviewed the literature to determine whether there was evidence for the effect of CKD on the renal secretion of basic drugs. We first screened 72 uremic solutes as inhibitors of [14C]-labeled metformin uptake by OCT2. Seven were identified as inhibitors and 3 of them were determined to be clinically relevant. Of the 7 solutes, dimethylamine, malondialdehyde, trimethylamine, homocysteine, indoxyl-β-d-glucuronide, and glutathione disulfide were novel OCT2 inhibitors. For 6 drugs that are known OCT2 substrates, both secretory clearance and glomerular filtration rate declined in parallel with progression of CKD from stage 2 to 4, suggesting that selective effects of uremic solutes on net tubular secretion of organic cations do not occur. Further clinical studies are warranted with a broader range of OCT2 substrates to determine whether CKD may differentially affect tubular secretion of drugs especially in patients with advanced CKD.

Oxalobacter formigenes colonization normalizes oxalate excretion in a gastric bypass model of hyperoxaluria

BackgroundHyperoxaluria and oxalate kidney stones frequently develop after Roux-en-Y gastric bypass (RYGB). Oxalobacter formigenes can degrade ingested oxalate. ObjectivesExamine the effect of O. formigenes wild rat strain (OXWR) colonization on urinary oxalate excretion and intestinal oxalate transport in a hyperoxaluric RYGB model. SettingBasic Science Laboratory, United States. MethodsAt 21 weeks of age, 28 obese male Sprague-Dawley rats survived Sham (n = 10) or RYGB (n = 18) surgery and were maintained on a 1.5% potassium oxalate, 40% fat diet. At 12 weeks postoperatively, half the animals in each group were gavaged with OXWR. At 16 weeks, percent dietary fat content was lowered to 10%. Urine and stool were collected weekly to determine oxalate and colonization status, respectively. At week 20, [14 C]-oxalate fluxes and electrical parameters were measured in vitro across isolated distal colon and jejunal (Roux limb) tissue mounted in Ussing Chambers. ResultsRYGB animals lost 22% total weight while Shams gained 5%. On a moderate oxalate diet, urinary oxalate excretion was 4-fold higher in RYGB than Sham controls. OXWR colonization, obtained in all gavaged animals, reduced urinary oxalate excretion 74% in RYGB and 39% in Sham and was further augmented by lowering the percentage of dietary fat. Finally, OXWR colonization significantly enhanced basal net colonic oxalate secretion in both groups. ConclusionsIn our model, OXWR lowered urinary oxalate by luminal oxalate degradation in concert with promotion of enteric oxalate elimination. Trials of O. formigenes colonization and low-fat diet are warranted in calcium oxalate stone formers with gastric bypass and resistant hyperoxaluria.

Pregnancy Increases the Renal Secretion of N1-methylnicotinamide, an Endogenous Probe for Renal Cation Transporters, in Patients Prescribed Metformin.

N1-methylnicotinamide (1-NMN) has been investigated as an endogenous probe for the renal transporter activity of organic cation transporter 2 (OCT2) and multidrug and toxin extrusion proteins 1 and 2-K (MATE1 and MATE2-K). As pregnancy increased the renal secretion of metformin, a substrate for OCT2, MATE1, and MATE2-K, we hypothesized that the renal secretion of 1-NMN would be similarly affected. Blood and urine samples collected from women prescribed metformin for type 2 diabetes, gestational diabetes, and polycystic ovarian syndrome during early, mid, and late pregnancy (n = 34 visits) and postpartum (n = 14 visits) were analyzed for 1-NMN using liquid chromatography-mass spectrometry. The renal clearance and secretion clearance, using creatinine clearance to correct for glomerular filtration, were estimated for 1-NMN and correlated with metformin renal clearance. 1-NMN renal clearance was higher in both mid (504 ± 293 ml/min, P < 0.01) and late pregnancy (557 ± 305 ml/min, P < 0.01) compared with postpartum (240 ± 106 ml/min). The renal secretion of 1-NMN was 3.5-fold higher in mid pregnancy (269± 267, P < 0.05) and 4.5-fold higher in late pregnancy compared with postpartum (342 ± 283 versus 76 ± 92 ml/min, P < 0.01). Because creatinine is also a substrate of OCT2, MATE1, and MATE2-K, creatinine clearance likely overestimates filtration clearance, whereas the calculated 1-NMN secretion clearance is likely underestimated. Metformin renal clearance and 1-NMN renal clearance were positively correlated (rs = 0.68, P < 0.0001). 1-NMN renal clearance increases during pregnancy due to increased glomerular filtration and net secretion by renal transporters.

Effects of maturity at ensiling of bermudagrass and fibrolytic enzyme application on the performance of early-lactation dairy cows

The objective of this study was to examine effects of adding fibrolytic enzymes to diets containing bermudagrass ensiled after 4 or 7wk of regrowth on the diet digestibility, ruminal fermentation and performance of lactating cows, and the interaction of the treatments. In experiment 1, 64 Holstein cows (22±4d in milk) were assigned to an experiment with a 2×2 factorial treatment arrangement and a 56-d duration. Treatments were diets containing 4 or 7wk regrowth bermudagrass silage without or with an exogenous fibrolytic enzyme cocktail. The cellulase-xylanase enzyme was applied at 2.33g/kg of total mixed ration dry matter (DM) during mixing immediately before feeding. Experiment 2 was aimed at examining treatment effects on the ruminal fermentation profile. Four ruminally cannulated cows were assigned to the 4 treatments using a 4×4 Latin square design with 14-d periods. No enzyme by maturity interaction was detected for any measurement. Regardless of forage maturity, applying the fibrolytic enzyme did not affect DM intake, milk yield, apparent digestibility, feed efficiency, energy balance, and ruminal fermentation though it tended to increase milk lactose concentration (4.88 vs. 4.81%). Feeding the 4-wk diet instead of the 7-wk diet increased DM intake (22.4 vs. 21.3kg/d), digestibility of DM, neutral detergent fiber, and acid detergent fiber, and tended to increase 3.5%-fat corrected milk yield (47.2 vs. 44.3kg/d) and milk fat yield (1.88 vs. 1.73kg/d). Therefore, daily intake of net energy and secretion of milk energy were greater for the 4-wk diet. In addition, the 4-wk diet increased the ruminal concentrations of acetate, propionate, valerate, lactate, and total volatile fatty acids, and decreased ruminal pH, without affecting the acetate:propionate ratio. Feeding fibrolytic enzymes did not improve the performance of early-lactation dairy cows, but harvesting the forage earlier tended to improve milk production.

Angiotensin II Type 1 Receptor-Dependent GLP-1 and PYY Secretion in Mice and Humans.

Angiotensin II (Ang II) is the key hormone mediator of the renin angiotensin system, which regulates blood pressure and fluid and electrolyte balance in the body. Here we report that in the colonic epithelium, the Ang II type 1 receptor is highly and exclusively expressed in enteroendocrine L cells, which produce the gut hormones glucagon-like peptide-1 and peptide YY (PYY). Ang II stimulated glucagon-like peptide-1 and PYY release from primary cultures of mouse and human colon, which was antagonized by the specific Ang II type 1 receptor blocker candesartan. Ang II raised intracellular calcium levels in L cells in primary cultures, recorded by live-cell imaging of L cells specifically expressing the fluorescent calcium sensor GCaMP3. In Ussing chamber recordings, Ang II reduced short circuit currents in mouse distal colon preparations, which was antagonized by candesartan or a specific neuropeptide Y1 receptor inhibitor but insensitive to amiloride. We conclude that Ang II stimulates PYY secretion, in turn inhibiting epithelial anion fluxes, thereby reducing net fluid secretion into the colonic lumen. Our findings highlight an important role of colonic L cells in whole-body fluid homeostasis by controlling water loss through the intestine.

Renal Transporter-Mediated Drug-Drug Interactions: Are They Clinically Relevant?

The kidney, through the distinct processes of passive glomerular filtration and active tubular secretion, plays an important role in the elimination of numerous endobiotics (eg, hormones, metabolites), toxins, nutrients, and drugs. Renal transport pathways mediating active tubular secretion and reabsorption in the proximal tubule are complex, involving apical and basolateral transporters acting in concert. Detailed studies of the molecular mechanisms of net active tubular secretion have established the involvement of multiple transporters with overlapping substrate specificity mediating competing secretion and reabsorption pathways. Although drug interactions arising from inhibition of renal transporters are rare relative to other mechanisms, they can involve commonly administered drugs (eg, cimetidine, metformin), may be underappreciated due to muted effects on plasma pharmacokinetics relative to tissue levels, can affect narrow-therapeutic-index medications (eg, antiarrhythmic, oncology medications), and may disproportionately affect sensitive populations where polypharmacy is common (eg, the elderly, diabetics). In particular, there is the potential for larger-magnitude interactions in subjects with reduced glomerular filtration rates due to the increased relative contribution of tubular secretion. The assessment of additional endpoints in drug-drug interaction studies including pharmacodynamics, positron emission tomography imaging, and metabolomics promises to expand our understanding of the clinical relevance of renal drug interactions.

Experimental diabetic diarrhea in rats. Intestinal mucosal denervation hypersensitivity and treatment with clonidine.

Diarrhea in streptozocin-induced chronically diabetic rats is caused by an impaired adrenergic regulation of intestinal fluid and electrolyte transport. Stimulation of alpha 2-adrenergic receptors on enterocytes normally promotes NaCl absorption and inhibits HCO3 secretion. The purpose of this study was to determine if adrenergic denervation of intestinal mucosa in chronically diabetic rats alters postsynaptic receptor response, and if the alpha 2-adrenergic agonist clonidine could correct observed fluid malabsorption. Mucosal norepinephrine stores, a measure of adrenergic tone, were markedly reduced in diabetic rats compared with nondiabetic littermates. In vitro, short-circuit current changes to exogenously added l-epinephrine were significantly greater in diabetics, suggesting that denervation hypersensitivity was due to increased numbers of postsynaptic alpha 2-adrenergic receptors. In vivo loop studies demonstrated net fluid secretion in the ileum and colon of diabetics. In diabetics, clonidine reversed the secretion to absorption, but it had no effect on fluid absorption in controls. We conclude that diabetic diarrhea in streptozocin-induced chronically diabetic rats is due to impaired adrenergic regulation of mucosal ion transport, accompanied by a postsynaptic denervation hypersensitivity that can be reversed by clonidine, and accompanied by net intestinal fluid secretion that can be effectively reversed with clonidine.

Pregnancy-related pharmacokinetic changes.

The pharmacokinetics of many drugs are altered by pregnancy. Drug distribution and protein binding are changed by pregnancy. While some drug metabolizing enzymes have an apparent increase in activity, others have an apparent decrease in activity. Not only is drug metabolism affected by pregnancy, but renal filtration is also increased. In addition, pregnancy alters the apparent activities of multiple drug transporters resulting in changes in the net renal secretion of drugs.

Do Compensatory Changes Occur in Intestinal Glucose Transport Following Gastric Bypass in Type 2, Diabetic Rats

Although there is a considerable amount of evidence in the literature showing that Rouen‐en‐Y‐Gastric Bypass (RYGB) in particular, is very effective in resolving Type 2, Diabetes (T2D), a very fundamental question that has not yet been addressed is how transmural glucose transport is modified across the newly configured anatomy of the upper intestinal segments following RYGB? Thus, the objective of the study was to examine potential adaptations in glucose transport across the Roux Limb and distal ileum of ZDF (Zucker Diabetic Fatty) and ZLC (Zucker Lean Control) rats following RYGBUnidirectional glucose fluxes were measured in Ussing chambers under symmetrical, short‐circuited conditions in vitro ~ 10 weeks following the surgical procedure. Segments that were removed included ~ 4 cm of proximal Roux Limb measured from the stomach pouch insertion point; a segment ~ 4 cm at the location measured 35 cm from the ileo‐caecal valve and representing the “predestined” Roux limb in control rats; and a similar size segment immediately proximal to the ileocaecal valve which is the most distal ileum that is not reconfigured in any animal. Using radio‐labeled [14C]‐3‐0‐methylglucose, unidirectional fluxes of glucose were measured across paired adjacent tissues from the mucosal to serosal bath, Jms (absorptive component) and in the opposite direction from serosal to mucosal, Jsm (secretory component) over a 45 min time period. The overall net transport of glucose (Jnet) was determined by subtracting the two unidirectional fluxes. Both the short‐circuit current (Isc) and the transepithelial voltage generated by the tissue is recorded at 15 min intervals and tissue conductance (GT) is calculated from these electrical parameters using Ohm's Law.Major changes in glucose fluxes occurred in obese ZDF/RYGB rats compared to ZDF controls in both of the intestinal segments examined. The unidirectional glucose fluxes are dramatically reduced in RYGB rats and the direction of net glucose transport is reversed across the Roux Limb. It is also notable that tissue conductance of the Roux Limb is reduced by ~ 48% (from GT of 12.0 ± 2.2 mS.cm‐2 in n=8 “predestined” Roux Limb tissues to GT = 6.2 ± 1.3 mS.cm‐2, n=6 Roux Limb tissues) consistent with the lowering (~40% in Jms and ~95% in Jsm) of both unidirectional glucose fluxes indicating an adaptive change in overall tissue permeability. A significant reduction (~82%) occurred in net glucose absorption across the ZDF/RYGB distal ileum (which is not surgically re‐configured) largely via a 64% reduction in Jms. The “predestined” Roux limb of the lean ZLC rats supports a very robust net absorption of glucose that is completely abolished in the reconfigured ZLC/RYGB limb and the unidirectional fluxes were greatly diminished (~90% in Jms and ~70% in Jsm) as was GT from 14.5 ± 2.8 mS.cm‐2 in n=4 “predestined” Roux Limb tissues to GT = 8.0 ± 1.1 mS.cm‐2, n=4 Roux Limb tissues) which is a very similar response to that observed in the obese ZDF rats. There were also major alterations in glucose fluxes across the distal ileum of the lean ZLC/RYGB rats which supported a robust net secretion compared to a net absorption in the lean ZLC controls and no change in GT. To our knowledge, these are the first measurements of intestinal glucose transport in ZDF rats with/without RYGB. In conclusion, we are proposing that altered intestinal handling of glucose may be contributing to the resolution of T2D post RYGB.Support or Funding InformationDK089000

Measuring intestinal fluid transport in vitro: Gravimetric method versus non-absorbable marker

The gut sac is a long-standing, widely used in vitro preparation for studying solute and water transport, and calculation of these fluxes requires an accurate assessment of volume. This is commonly determined gravimetrically by measuring the change in mass over time. While convenient this likely under-estimates actual net water flux (Jv) due to tissue edema. We evaluated whether the popular in vivo volume marker [14C]-PEG 4000, offers a more representative measure of Jvin vitro. We directly compared these two methods in five teleost species (toadfish, flounder, rainbow trout, killifish and tilapia). Net fluid absorption by the toadfish intestine based on PEG was significantly higher, by almost 4-fold, compared to gravimetric measurements, compatible with the latter under-estimating Jv. Despite this, PEG proved inconsistent for all of the other species frequently resulting in calculation of net secretion, in contrast to absorption seen gravimetrically. Such poor parallelism could not be explained by the absorption of [14C]-PEG (typically <1%). We identified a number of factors impacting the effectiveness of PEG. One was adsorption to the surface of sample tubes. While it was possible to circumvent this using unlabelled PEG 4000, this had a deleterious effect on PEG-based Jv. We also found sequestration of PEG within the intestinal mucus. In conclusion, the short-comings associated with the accurate representation of Jv by gut sac preparations are not overcome by [14C]-PEG. The gravimetric method therefore remains the most reliable measure of Jv and we urge caution in the use of PEG as a volume marker.