Peter Geibel

The calcium sensing receptor modulates fluid reabsorption and acid secretion in the proximal tubule

The proximal tubule uses a complex process of apical acid secretion and basolateral bicarbonate absorption to regulate both luminal acidification and fluid absorption. One of the primary regulators of apical acid secretion is the luminal sodium-hydrogen exchanger expressed along the apical membrane of the proximal tubule. Similarly, the calcium-sensing receptor (CaSR) is also located along the luminal membrane of the proximal tubule. Here we investigated the role of CaSR in proton secretion and fluid reabsorption in proximal tubules by modulating luminal calcium concentration, using both in vivo micropuncture in rats and in vitro perfused mouse proximal tubules. Using CaSR knockout mice and a calcimimetic agent, we found that increased proton secretion and fluid reabsorption were CaSR dependent. Activating CaSR by either raising the luminal calcium ion concentration or by the calcimimetic caused a concomitant increase in sodium-dependent proton extrusion and fluid reabsorption, whereas in proximal tubules isolated from CaSR knockout mice varying calcium ion concentration had no effect. Application of a calcimimetic in lower concentrations of calcium ion stimulated these processes in vitro and in vivo. Thus, in both rats and mice, increased luminal calcium concentration leads to enhanced fluid reabsorption in the proximal tubule, a process related to activation of CaSR.

Functional vacuolar ATPase (V-ATPase) proton pumps traffic to the enterocyte brush border membrane and require CFTR

Vacuolar ATPases (V-ATPases) are highly conserved proton pumps that regulate organelle pH. Epithelial luminal pH is also regulated by cAMP-dependent traffic of specific subunits of the V-ATPase complex from endosomes into the apical membrane. In the intestine, cAMP-dependent traffic of cystic fibrosis transmembrane conductance regulator (CFTR) channels and the sodium hydrogen exchanger (NHE3) in the brush border regulate luminal pH. V-ATPase was found to colocalize with CFTR in intestinal CFTR high expresser (CHE) cells recently. Moreover, apical traffic of V-ATPase and CFTR in rat Brunners glands was shown to be dependent on cAMP/PKA. These observations support a functional relationship between V-ATPase and CFTR in the intestine. The current study examined V-ATPase and CFTR distribution in intestines from wild-type, CFTR(-/-) mice and polarized intestinal CaCo-2BBe cells following cAMP stimulation and inhibition of CFTR/V-ATPase function. Coimmunoprecipitation studies examined V-ATPase interaction with CFTR. The pH-sensitive dye BCECF determined proton efflux and its dependence on V-ATPase/CFTR in intestinal cells. cAMP increased V-ATPase/CFTR colocalization in the apical domain of intestinal cells and redistributed the V-ATPase Voa1 and Voa2 trafficking subunits from the basolateral membrane to the brush border membrane. Voa1 and Voa2 subunits were localized to endosomes beneath the terminal web in untreated CFTR(-/-) intestine but redistributed to the subapical cytoplasm following cAMP treatment. Inhibition of CFTR or V-ATPase significantly decreased pHi in cells, confirming their functional interdependence. These data establish that V-ATPase traffics into the brush border membrane to regulate proton efflux and this activity is dependent on CFTR in the intestine.

Effect of NSAIDs on Na+/H+ exchanger activity in rat colonic crypts

Nonsteroidal anti-inflammatory drugs (NSAIDs; 1) are widely recommended for several acute and chronic conditions. For example, both indomethacin and aspirin are taken for pain relief. Aspirin is also used for prevention of myocardial infarction, and indomethacin can be administered orally or as a suppository for patients with rheumatoid disease and other chronic inflammatory states. However, use of NSAIDs can cause damage to the mucosal barrier surrounding the gastrointestinal (GI) tract, increasing the risk of ulcer formation. While microencapsulation of NSAIDs has been shown to reduce upper GI injury, sustained release in the lower GI tract and colon may cause epithelial erosion due to increased acidification. The use of suppositories has also been linked to rectal and lower GI bleeding. In this study, we investigated the role of NSAIDs aspirin and indomethacin on Na⁺/H⁺ exchanger (NHE) activity in rat colonic crypts. By comparing average rates of pH recovery between control and NSAID perfusion runs, we were able to determine that both aspirin and indomethacin increase hydrogen extrusion into the colonic lumen. Through treatment with 5-ethylisopropyl amiloride (EIPA), amiloride, and zoniporide dihydrochloride, we further demonstrated that indomethacin specifically enhances proton excretion through regulation of apical NHE-3 and NHE-2 and to a lesser extent on basolateral NHE-1 and NHE-4. Our results suggest that clinical exposure to NSAIDs may affect colonic tissue at the site of selected NHE isoforms, resulting in modulation of transport and barrier function.

Identification of Functionally Distinct Na-HCO3 Co-Transporters in Colon

Na-HCO3 cotransport (NBC) regulates intracellular pH (pHi) and HCO3 secretion in rat colon. NBC has been characterized as a 5,5′-diisothiocyanato-2-2′-stilbene (DIDS)-sensitive transporter in several tissues, while the colonic NBC is sensitive to both amiloride and DIDS. In addition, the colonic NBC has been identified as critical for pHi regulation as it is activated by intravesicular acid pH. Molecular studies have identified several characteristically distinct NBC isoforms [i.e. electrogenic (NBCe) and electroneutral (NBCn)] that exhibit tissue specific expression. This study was initiated to establish the molecular identity and specific function of NBC isoforms in rat colon. Northern blot and reverse transcriptase PCR (RT-PCR) analyses revealed that electrogenic NBCe1B or NBCe1C (NBCe1B/C) isoform is predominantly expressed in proximal colon, while electroneutral NBCn1C or NBCn1D (NBCn1C/D) is expressed in both proximal and distal colon. Functional analyses revealed that amiloride-insensitive, electrogenic, pH gradient-dependent NBC activity is present only in basolateral membranes of proximal colon. In contrast, amiloride-sensitive, electroneutral, [H+]-dependent NBC activity is present in both proximal and distal colon. Both electrogenic and electroneutral NBC activities are saturable processes with an apparent Km for Na of 7.3 and 4.3 mM, respectively; and are DIDS-sensitive with apparent Ki of 8.9 and 263.8 µM, respectively. In addition to Na-H exchanger isoform-1 (NHE1), pHi acidification is regulated by a HCO3-dependent mechanism that is HOE694-insensitive in colonic crypt glands. We conclude from these data that electroneutral, amiloride-sensitive NBC is encoded by NBCn1C/D and is present in both proximal and distal colon, while NBCe1B/C encodes electrogenic, amiloride-insensitive Na-HCO3 cotransport in proximal colon. We also conclude that NBCn1C/D regulates HCO3-dependent HOE694-insensitive Na-HCO3 cotransport and plays a critical role in pHi regulation in colonic epithelial cells.

Generating vascular conduits: from tissue engineering to three-dimensional bioprinting

Abstract Vascular disease – including coronary artery disease, carotid artery disease, and peripheral vascular disease – is a leading cause of morbidity and mortality worldwide. The standard of care for restoring patency or bypassing occluded vessels involves using autologous grafts, typically the saphenous veins or internal mammary arteries. Yet, many patients who need life- or limb-saving procedures have poor outcomes, and a third of patients who need vascular intervention have multivessel disease and therefore lack appropriate vasculature to harvest autologous grafts from. Given the steady increase in the prevalence of vascular disease, there is great need for grafts with the biological and mechanical properties of native vessels that can be used as vascular conduits. In this review, we present an overview of methods that have been employed to generate suitable vascular conduits, focusing on the advances in tissue engineering methods and current three-dimensional (3D) bioprinting methods. Tissue-engineered vascular grafts have been fabricated using a variety of approaches such as using preexisting scaffolds and acellular organic compounds. We also give an extensive overview of the novel use of 3D bioprinting as means of generating new vascular conduits. Different strategies have been employed in bioprinting, and the use of cell-based inks to create de novo structures offers a promising solution to bridge the gap of paucity of optimal donor grafts. Lastly, we provide a glimpse of our work to create scaffold-free, bioreactor-free, 3D bioprinted vessels from a combination of rat vascular smooth muscle cells and fibroblasts that remain patent and retain the tensile and mechanical strength of native vessels.

S2063 Calcium Sensing Receptor (CaSR) Function in Intestine Epithelial Cells of Rodents and Humans

Membranous calcium-sensing receptors (CaSR) on intestinal epithelial cells are difficult to study, due to fast loss of function following isolation of the cells. We established a chemical isolation process to maintain receptor function, being able to measure receptor-activity in a fluorescence spectrometry setup. In diarrhea, high intracellular concentrations of cyclic nucleotides in intestine epithelial cells lead to net fluid excretion due to enhanced serosal to mucosal fluid-transport. We showed earlier the reversibility of this effect via activation of CaSR with calcimimetic agents including natural and synthetic small molecules such as the compound R-568. Exposure to these allosteric modifiers in the presence of calcium resulted in a cessation of fluid excretion, and enhanced absorption. In the present study we develop a new isolation method for single cells of all intestinal segments and demonstrate that CaSR remains viable and active upon stimulationwith allostericmodifiers or by increasing the extracellular calcium concentration. These results give an important new tool for intestinal transport screening. METHODS We generated functional individual intestinal epithelial cells from the duodenum, jejunum, ileum and colon of female rats and human colon, using EDTA digestion solution and light mechanical force at 37°C for 20 minutes. Cells were loaded with 2nM Fluo-4 and Fluo-8, calcium-sensing fluorescent dyes. After multiple washout-steps and sedimentation for two hours, the activity of the CaSRwas determined, using a fluorescence spectrometer measuring the fluorescent excitation at 516nm. RESULTS We were able to show the functionality of epithelial CaSR on epithelial cells from four sections of the intestine of the rat: duodenum, jejunum, ileum and colon, and also human colon. The CaSR shows a dose dependent activation over a concentration range of 0.125mM to 2.5mM. The EC50 for calcium for the freshly isolated cells was comparable to that previously obtained for intact native tissue. In a separate study isolated cells were exposed to R-568 prior to the calcium dose curve, resulting in a left phase shift of the activation curve indicative of enhanced calcium binding to the receptor. CONCLUSION Our studies demonstrate that it is now possible to develop viable isolated cells from the entire digestive tract that maintain CaSR density. This technique provides an important new screening tool for use in testing pharmacological agents on intestinal epithelia.