Ammonia metabolism is important in multiple aspects of gastrointestinal physiology, but the mechanisms of ammonia transport in the gastrointestinal tract remain incompletely defined. The present study examines expression of the ammonia transporter family members Rh B glycoprotein (RhBG) and Rh C glycoprotein (RhCG) in the mouse gastrointestinal tract. Real-time RT-PCR amplification and immunoblot analysis identified mRNA and protein for both RhBG and RhCG were expressed in stomach, duodenum, jejunum, ileum, and colon. Immunohistochemistry showed organ and cell-specific expression of both RhBG and RhCG. In the stomach, both RhBG and RhCG were expressed in the fundus and forestomach, but not in the antrum. In the forestomach, RhBG was expressed by all nucleated squamous epithelial cells, whereas RhCG was expressed only in the stratum germinativum. In the fundus, RhBG and RhCG immunoreactivity was present in zymogenic cells but not in parietal or mucous cells. Furthermore, zymogenic cell RhBG and RhCG expression was polarized, with apical RhCG and basolateral RhBG immunoreactivity. In the duodenum, jejunum, ileum, and colon, RhBG and RhCG immunoreactivity was present in villous, but not in mucous or crypt cells. Similar to the fundic zymogenic cell, RhBG and RhCG expression in villous epithelial cells was polarized when apical RhCG and basolateral RhBG immunoreactivity was present. Thus the ammonia transporting proteins RhBG and RhCG exhibit cell-specific, axially heterogeneous, and polarized expression in the intestinal tract suggesting they function cooperatively to mediate gastrointestinal tract ammonia transport.

The renal collecting duct is the primary site for the ammonia secretion necessary for acid-base homeostasis. Recent studies have identified the presence of putative ammonia transporters in the collecting duct, but whether the collecting duct has transporter-mediated ammonia transport is unknown. The purpose of this study was to examine basolateral ammonia transport in the mouse collecting duct cell (mIMCD-3). To examine mIMCD-3 basolateral ammonia transport, we used cells grown to confluence on permeable support membranes and quantified basolateral uptake of the radiolabeled ammonia analog [14C]methylammonia ([14C]MA). mIMCD-3 cell basolateral MA transport exhibited both diffusive and transporter-mediated components. Transporter-mediated uptake exhibited a Km for MA of 4.6 +/- 0.2 mM, exceeded diffusive uptake at MA concentrations below 7.0 +/- 1.8 mM, and was competitively inhibited by ammonia with a Ki of 2.1 +/- 0.6 mM. Transporter-mediated uptake was not altered by inhibitors of Na+-K+-ATPase, Na+-K+-2Cl(-) cotransporter, K+ channels or KCC proteins, by excess potassium, by extracellular sodium or potassium removal or by varying membrane potential, suggesting the presence of a novel, electroneutral ammonia-MA transport mechanism. Increasing the outwardly directed transmembrane H+ gradient increased transport activity by increasing Vmax. Finally, mIMCD-3 cells express mRNA and protein for the putative ammonia transporter Rh B-glycoprotein (RhBG), and they exhibit basolateral RhBG immunoreactivity. We conclude that mIMCD-3 cells express a basolateral electroneutral NH4+/H+ exchange activity that may be mediated by RhBG.

The mechanism of glomerular infiltration of monocytes remains unknown in diabetic nephropathy. We examined the effect of a high glucose concentration on monocyte chemotactic peptide 1 (MCP-1) expression in human mesangial cells (MCs) by using enzyme-linked immunosorbent assay and reverse transcription coupled with polymerase chain reaction (PCR). More than a 50% increase in the MCP-1 protein production was observed in MCs cultured in high-glucose medium (450 mg/dl) as compared to normal glucose (100 mg/dl; 1,496 ± 75 vs. 966 ± 15 pg/ml after 24 h, 1,910 ± 93 vs. 1,250 ± 55 pg/ml after 48 h). Semiquantitative PCR showed that phorbol myristate acetate (100 nM) increased the ratio of PCR products for MCP-1 to housekeeping gene glyceraldehyde-3-phosphate dehydrogenase on densitometric results at 24 h by 2.7-fold, which was prevented by calphostin C (200 nM) pretreatment. High glucose increased the ratio by 3-fold as compared to normal glucose at 24 h (0.72 ± 0.11 vs. 0.24 ± 0.01). This was also suppressed by calphostin C pretreatment. These findings demonstrate that high glucose can directly increase MCP-1 expression in MCs, which may contribute to monocyte infiltration in diabetic nephropathy, and this is regulated by protein kinase C.

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Handlogten, Mary E., Seong-Pyo Hong, Connie M. Westhoff, and I. David Weiner. Apical ammonia transport by the mouse inner medullary collecting duct cell (mIMCD-3).