Experimental Colitis Is Associated with Transcriptional Inhibition of Na+/Ca2+ Exchanger Isoform 1 (NCX1) Expression by Interferon γ in the Renal Distal Convoluted Tubules

NCX1 is a Na⁺/Ca²⁺ exchanger, which is believed to provide a key route for basolateral Ca²⁺ efflux in the renal epithelia, thus contributing to renal Ca²⁺ reabsorption. Altered mineral homeostasis, including intestinal and renal Ca²⁺ transport may represent a significant component of the pathophysiology of the bone mineral density loss associated with Inflammatory Bowel Diseases (IBD). The objective of our research was to investigate the effects of TNBS and DSS colitis and related inflammatory mediators on renal Ncx1 expression. Colitis was associated with decreased renal Ncx1 expression, as examined by real-time RT-PCR, Western blotting, and immunofluorescence. In mIMCD3 cells, IFNγ significantly reduced Ncx1 mRNA and protein expression. Similar effects were observed in cells transiently transfected with a reporter construct bearing the promoter region of the kidney-specific Ncx1 gene. This inhibitory effect of IFNγ is mediated by STAT1 recruitment to the proximal promoter region of Ncx1. Further in vivo study with Stat1^−/− mice confirmed that STAT1 is indeed required for the IFNγ mediated Ncx1 gene regulation. These results strongly support the hypothesis that impaired renal Ca²⁺ handling occurs in experimental colitis. Negative regulation of NCX1- mediated renal Ca²⁺ absorption by IFNγ may significantly contribute to the altered Ca²⁺ homeostasis in IBD patients and to IBD-associated loss of bone mineral density.     

Molecular cloning of a murine type III sodium-dependent phosphate cotransporter (Pit-2) gene promoter.

We report the novel cloning and preliminary characterization of a murine type III sodium-dependent phosphate cotransporter (Pit-2) gene promoter. Five promoter/luciferase reporter gene constructs, -1816/+61, -1620/+61, -1223/+61, -600/+61 and -225/+61, showed significant luciferase activity (6-14-fold over background) when transfected into human colon carcinoma (Caco-2) and opossum kidney (OKP) cells.     

Glutamine transport by rat basolateral membrane vesicles

Glutamine, a neutral amino acid, is unlike most amino acids, has two amine moieties which underlies its importance as a nitrogen transporter and a carrier of ammonia from the periphery to visceral organs. The gastrointestinal tract utilizes glutamine as a respiratory substrate. The intestinal tract receives glutamine from the luminal side and from the arterial side through the basolateral membranes of the enterocyte. This study characterizes the transport of glutamine by basolateral membrane vesicles of the rat. Basolateral membranes were prepared by a well validated technique of separation on a percoll density gradient. Membrane preparations were enriched with Na+/K+-ATPase and showed no 'overshoot' phenomena with glucose under sodium-gradient conditions. Glutamine uptake represented transport into the intravesicular space as evident by an osmolality study. Glutamine uptake was temperature sensitive and driven by an inwardly directed sodium gradient as evident by transient accumulation of glutamine above the equilibrium values. Kinetics of glutamine uptake under both sodium and potassium gradients at glutamine concentrations between 0.01 and 0.6 mM showed saturable processes with Vmax of 0.39 +/- 0.008 and 0.34 +/- 0.05 nmol/mg protein per 15 s for both sodium-dependent and sodium-independent processes, respectively. Km values were 0.2 +/- 0.01 and 0.55 +/- 0.01 mM, respectively. pH optimum for glutamine uptake was 7.5. Imposition of negative membrane potential by valinomycin and anion substitution studies enhanced the sodium-dependent uptake of glutamine suggesting an electrogenic process, whereas the sodium-independent uptake was not enhanced suggesting an electroneutral process. Other neutral amino acids inhibited the initial uptake of glutamine under both sodium-dependent and sodium-independent conditions. We conclude that glutamine uptake by basolateral membranes occurs by carrier-mediated sodium-dependent and sodium-independent processes. Both processes exhibit saturation kinetics and are inhibited by neutral amino acids. The sodium-dependent pathway is electrogenic whereas the sodium-independent pathway is electroneutral.     

Chloride transport across rat ileal basolateral membrane vesicles.

The present study was designed to investigate Cl- transport across rat ileal basolateral membranes. Basolateral membrane vesicles were prepared by a well-validated technique. The purity of the basolateral membrane vesicles was verified by marker enzyme studies and by studies of d-glucose and calcium uptake. Cl- uptake was studied by a rapid filtration technique. Neither an outwardly directed pH gradient, nor a HCO3- gradient, or their combination could elicit any stimulation of Cl- transport when compared with no gradient. 4,4-Diisothiocyanostilbene-2,2-disulfonic acid at 5 mM concentration did not inhibit Cl- uptake under gradient condition. Similarly, the presence of the combination of outwardly directed Na+ and HCO3- gradients did not stimulate Cl- uptake compared with the combination of K+ and HCO3- gradients or no HCO3- gradient. This is in contrast to our results in the brush border membranes, where an outwardly directed pH gradient caused an increase in Cl- uptake. Cl- uptake was stimulated in the presence of combined Na+ and K+ gradient. Bumetanide at 0.1 mM concentration inhibited the initial rate of Cl- uptake in the presence of combined Na+ and K+ gradients. Kinetic studies of bumetanide-sensitive Cl- uptake showed a Vmax of 5.6 +/- 0.7 nmol/mg protein/5 sec and a Km of 30 +/- 8.7 mM. Cl- uptake was stimulated by an inside positive membrane potential induced by the ionophore valinomycin in the setting of inwardly directed K+ gradient compared with voltage clamp condition. These studies demonstrate two processes for Cl- transport across the rat ileal basolateral membrane: one is driven by an electrogenic diffusive process and the second is a bumetanide-sensitive Na+/K+/2 Cl- process. Cl- uptake is not enhanced by pH gradient, HCO3- gradient, their combination, or outwardly directed HCO3- and Na+ gradients.     

Intestinal calcium transport in spontaneously hypertensive rats (SHR) and their genetically matched WKY rats

Calcium transport across the basolateral membranes of the enterocyte represents the active step in calcium translocation. This step occurs by two mechanisms, an ATP-dependent pump and a Ca2+/Na+ exchange process. These studies were designed to investigate these two processes in jejunal basolateral membrane vesicles (BLMV) of the spontaneously hypertensive rats (SHR) and their genetically matched controls, Wistar-Kyoto (WKY) rats. The ATP-dependent calcium uptake was stimulated several-fold compared with no ATP condition in both SHR and WKY, but no differences were noted between rate of calcium uptake in SHR and WKY. Kinetics of ATP-dependent calcium uptake at concentrations between 0.01 and 1.0 microM revealed a Vmax of 0.67 +/- 0.03 nmol/mg protein/20 sec and a Km of 0.2 +/- 0.03 microM in SHR and Vmax of 0.69 +/- 0.12 and a Km of 0.32 +/- 0.14 microM in WKY rats. Ca2+/Na+ exchange in jejunal BLMV of SHR and WKY was investigated in two ways. First, sodium was added to the incubation medium (cis-Na+). Second, Ca2+ efflux from BLMV was studied in the presence of extravesicular Na+ (trans-Na+). Both studies suggest a decreased exchange of calcium and Na+. Kinetic parameters of Na(+)-dependent Ca2+ uptake at concentrations between 0.01 and 1.0 microM exhibited Vmax of 0.05 +/- 0.01 nanmol/mg protein/5 sec and a Km of 0.21 +/- 0.13 microM in SHR and Vmax of 0.11 +/- 0.02 nanmol/mg protein/5 sec and a Km of 0.09 +/- 0.05 in WKY, respectively. These results confirm that the intestinal BLMV of SHR and WKY rats have two mechanisms for calcium extrusion, an ATP-dependent Ca2+ transport process and a Na+/Ca2+ exchange process. The ATP-dependent process appears to be functional in SHR; however, the Ca2+/Na+ exchange mechanism appears to have a marked decrease in its maximal capacity. These findings suggest that calcium extrusion via Ca2+/Na+ is impaired in the SHR, which may lead to an increase in intracellular calcium concentration. These findings may have relevance to the development of hypertension.     

Effect of angiotensin-II on renal Na+/H+ exchanger-NHE3 and NHE2

The purpose of the present study was to determine the effect of angiotensin II (A-II) on membrane expression of Na+/H+ exchange isoforms NHE3 and NHE2 in the rat renal cortex. A-II (500 ng/kg per min) was chronically infused into the Sprague-Dawley rats by miniosmotic pump for 7 days. Arterial pressure and circulating plasma A-II level were significantly increased in A-II rats as compared to control rats. pH-dependent uptake of 22Na+ study in the presence of 50 microM HOE-694 revealed that Na+ uptake mediated by NHE3 was increased approximately 88% in the brush border membrane from renal cortex of A-II-treated rats. Western blotting showed that A-II increased NHE3 immunoreactive protein levels in the brush border membrane of the proximal tubules by 31%. Northern blotting revealed that A-II increased NHE3 mRNA abundance in the renal cortex by 42%. A-II treatment did not alter brush border NHE2 protein abundance in the renal proximal tubules. In conclusion, chronic A-II treatment increases NHE3-mediated Na+ uptake by stimulating NHE3 mRNA and protein content.     

Reduced Epithelial Na+/H+ Exchange Drives Gut Microbial Dysbiosis and Promotes Inflammatory Response in T Cell-Mediated Murine Colitis

Inflammatory bowel diseases (IBD) are associated with functional inhibition of epithelial Na⁺/H⁺ exchange. In mice, a selective disruption of NHE3 (Slc9a3), a major apical Na⁺/H⁺ exchanger, also promotes IBD-like symptoms and gut microbial dysbiosis. We hypothesized that disruption of Na⁺/H⁺ exchange is necessary for the development of dysbiosis, which promotes an exacerbated mucosal inflammatory response. Therefore, we performed a temporal analysis of gut microbiota composition, and mucosal immune response to adoptive T cell transfer was evaluated in Rag2^-/- and NHE3^-/-/Rag2^-/- (DKO) mice with and without broad-spectrum antibiotics. Microbiome (16S profiling), colonic histology, T cell and neutrophil infiltration, mucosal inflammatory tone, and epithelial permeability were analyzed. In adoptive T cell transfer colitis model, Slc9a3 status was the most significant determinant of gut microbial community. In DKO mice, NHE3-deficiency and dysbiosis were associated with dramatically accelerated and exacerbated disease, with rapid body weight loss, increased mucosal T cell and neutrophil influx, increased mucosal cytokine expression, increased permeability, and expansion of CD25^-FoxP3⁺ Tregs; this enhanced susceptibility was alleviated by oral broad-spectrum antibiotics. Based on these results and our previous work, we postulate that epithelial electrolyte homeostasis is an important modulator in the progression of colitis, acting through remodeling of the gut microbial community.