A Bifidobacterium probiotic strain and its soluble factors alleviate chloride secretion by human intestinal epithelial cells.

Previous studies indicate that certain probiotic bacterial strains or their soluble products can alleviate proinflammatory cytokine secretion by intestinal epithelial cells (IEC), but their impact on epithelial chloride (Cl(-)) secretion remains elusive. To further decipher the mechanisms of the cross-talk between bacteria/soluble factors and epithelial cells, we analyzed the capacity of the probiotic strain Bifidobacterium breve C50 (Bb C50), its conditioned medium, and other commensal Gram (+) bacteria to modulate epithelial Cl(-) secretion. The effect of Bb C50 on carbachol- (CCh) or forskolin (Fsk)-induced Cl(-) secretion was measured in an IEC line in Ussing chambers. The mechanisms involved in the regulation of Cl(-) secretion were assessed by measuring intracellular Ca(2+) concentration, phosphatase activity, protein kinase (PK) C and PKA activation, and cystic fibrosis transmembrane conductance regulator (CFTR) expression. CCh- or Fsk-induced Cl(-) secretion [short-circuit current (Isc): 151 +/- 28 and 98 +/- 14 microA/cm(2), respectively] was inhibited dose-dependently by Bb C50 (Isc 33 +/- 12 and 49 +/- 7 microA/cm(2) at multiplicity of infection 100; P < 0.02). Fsk-induced Cl(-) secretion was also inhibited by Lactobacillus rhamnosus 10893. No other inhibitory effect was recorded with the other Gram (+) bacteria tested. The inhibitory effect of Bb C50 on CCh-induced Cl(-) secretion targeted a step downstream of epithelial Ca(2+) mobilization and was associated with decreased PKC activity. Thus, Bb C50 and secreted soluble factors, by inhibiting phosphorylation processes, may promote intestinal homeostasis by controlling Cl(-) secretion.

Regulation of ROMK (Kir 1.1) channel expression in kidney thick ascending limb by hypertonicity: role of TonEBP and MAPK pathways.

The present study assessed the mechanisms by which hypertonicity caused by NaCl enhances the renal outer medullary potassium channel (ROMK) mRNA abundance in rat kidney medullary thick ascending limb (MTAL) and in cultured mouse TAL cells. Using the run-off technique, we observed that the ROMK gene transcription rate in nuclei isolated from MTAL fragments was enhanced approximately 40% by a high NaCl medium. In MTAL fragments, hypertonicity (450 mosm) caused by NaCl, not by mannitol or urea, enhanced both ROMK mRNA abundance and tonicity-responsive enhancer binding protein (TonEBP) total abundance and nuclear localization. In an immortalized mouse TAL cell culture in which ROMK is apically expressed, hypertonicity caused by both NaCl and mannitol, not urea, enhanced both ROMK mRNA abundance and TonEBP total abundance and nuclear localization. Confocal microscopy confirmed an increased nuclear translocation of TonEBP in response to NaCl-induced hypertonicity. Finally, inhibition of the p38 MAPK pathway by SB203580 and of the ERK pathway by PD98059 abolished the NaCl-induced stimulation of TonEBP and ROMK. These results establish that mRNA expression of ROMK is augmented in the MTAL by NaCl-induced hypertonicity through stimulation of ROMK gene transcription, and that TonEBP and the p38 MAPK and ERK pathways are involved in this effect.

Renal handling of NH3/NH4+: recent concepts.

To be appropriately excreted in urine, NH4+, the major component of urinary acid excretion, must be synthesized by proximal tubular cells, secreted into the proximal tubular fluid, reabsorbed by the medullary thick ascending limb (MTAL) to be accumulated in the medullary interstitium, and finally secreted in medullary collecting ducts. Several targets have been identified to account at the gene expression level for the adaptation of renal NH4+ synthesis and transport in response to a chronic acid load. These targets are the key enzymes of ammoniagenesis (mitochondrial glutaminase and glutamate dehydrogenase) and gluconeogenesis (phosphoenolpyruvate carboxykinase) and the Na+/H+(NH4+) exchanger NHE3 in the proximal tubule, the apical Na+-K+(NH4+)-2Cl- cotransporter of the MTAL, the basolateral Na+-K+(NH4+)-2Cl- cotransporter, and likely the epithelial Rh B and C glycoproteins in the collecting ducts. An acid pH per se appears to be a major factor in the control of the expression of these genes during metabolic acidosis probably through activation of pH sensors. Glucocorticoids may also act in concert with an acid pH to coordinate the adaptation of various tubular cell types. The present review focuses on some new aspects of NH3/ NH4+ transport and of regulations of gene expression that have recently emerged.

Renal handling of NH4+ in relation to the control of acid-base balance by the kidney.

The major component of urinary acid excretion is NH4+. To be appropriately excreted in urine, NH4+ must be synthesized by proximal tubular cells, secreted into the proximal tubular fluid, reabsorbed by the medullary thick ascending limb (MTAL) to be accumulated in the medullary interstitium, and finally secreted in medullary collecting ducts. Each step of this renal pathway is highly regulated and, in addition to acute events mediated by peptide hormones such as parathyroid hormone, the control of gene expression explains how the renal handling of NH4+ fully adapts to chronic changes in the acid-base status. Several targets have been identified at the gene expression level to account for the adaptation of renal NH4+ synthesis and transport in response to an acid load. These are the key enzymes of ammoniagenesis (mitochondrial glutaminase and glutamate dehydrogenase) and gluconeogenesis (phosphoenolpyruvate carboxykinase) in the proximal tubule, the apical Na(+)-K+(NH4+)-2Cl- cotransporter of the MTAL, and the basolateral Na(+)-K+(NH4+)-2Cl- cotransporter of medullary collecting ducts. At least two factors control the expression of these genes during metabolic acidosis: an acid pH and glucocorticoids, which appear to act in concert to coordinate the adaptation of various tubular cell types. The present review focuses on some aspects of these regulations that have been recently elucidated.

Increased activity of the diastrophic dysplasia sulfate transporter in otosclerosis and its inhibition by sodium fluoride.

HYPOTHESIS: This study investigates the function of the diastrophic dysplasia sulfate transporter (DTDST) in otosclerotic bone and the effect on it of sodium fluoride (NaF). BACKGROUND: Otosclerosis is a localized bone dystrophy with increased bone turnover. DTDST is implicated in the regulation of the bone turnover. MATERIALS AND METHODS: Primary cultures of cells were obtained from the stapes and external auditory canal (EAC) of 26 patients with otosclerosis and from nine control patients. Sulfate uptake was quantified under basal conditions and with NaF. The NaF signaling pathways were investigated using forskolin and verapamil. RESULTS: The relative initial rates of sulfate uptake and the apparent Vmax values were: otosclerotic stapes > EAC > control stapes = control EAC. The sulfate uptake by the otosclerotic stapes was correlated with the loss of sensorineural hearing. The amounts of DTDST mRNA (RNase protection assay) in the four subgroups did not differ. NaF (10(-6)M, 1 hr) inhibited sulfate uptake by the otosclerotic stapes and EAC cells but not by control samples. CONCLUSION: The authors believe that whether the increased DTDST activity is a cause or an effect of otosclerosis, it appears to be a specific target for NaF treatment.

Exploring the role of galectin 3 in kidney function: a genetic approach.

Galectin 3 belongs to a family of glycoconjugate-binding proteins that participate in cellular homeostasis by modulating cell growth, adhesion, and signaling. We studied adult galectin 3 null mutant (Gal 3-/-) and wild-type (WT) mice to gain insights into the role of galectin 3 in the kidney. By immunofluorescence, galectin 3 was found in collecting duct (CD) principal and intercalated cells in some regions of the kidney, as well as in the thick ascending limbs at lower levels. Compared to WT mice, Gal 3-/- mice had approximately 11% fewer glomeruli (p < 0.04), associated with kidney hypertrophy (p < 0.006). In clearance experiments, urinary chloride excretion was found to be higher in Gal 3-/- than in WT mice (p < 0.04), but there was no difference in urinary bicarbonate excretion, in glomerular filtration, or urinary flow rates. Under chronic low sodium diet, Gal 3-/- mice had lower extracellular fluid (ECF) volume than WT mice (p < 0.05). Plasma aldosterone concentration was higher in Gal 3-/- than in WT mice (p < 0.04), which probably caused the observed increase in alpha-epithelial sodium channel (alpha-ENaC) protein abundance in the mutant mice (p < 0.001). Chronic high sodium diet resulted paradoxically in lower blood pressure (p < 0.01) in Gal 3-/- than in WT. We conclude that Gal 3-/- mice have mild renal chloride loss, which causes chronic ECF volume contraction and reduced blood pressure levels.

Acid pH increases the stability of BSC1/NKCC2 mRNA in the medullary thick ascending limb.

Chronic metabolic acidosis enhances the ability of the medullary thick ascending limb (MTAL) to absorb NH(4)(+) at least in part by stimulating the mRNA and protein expression of BSC1/NKCC2, the MTAL apical Na(+)-K(+)(NH(4)(+))-2Cl(-) co-transporter. For assessing the mechanism by which an acid pH enhances the BSC1 mRNA abundance, MTAL were harvested from adrenalectomized rats and incubated in control (pH 7.35) and acid (pH 7.10) 1:1 mixtures of Ham's nutrient mixture F-12 and DME. rBSC1 mRNA abundance and gene transcription rate were quantified by quantitative reverse transcription-PCR and run-off assay, respectively. Acid incubation enhanced mRNA abundance within 4 h in whole cell (P < 0.02) but not in nucleus. BSC1 gene transcription rate was not affected by acid incubation. In contrast, under conditions in which gene transcription was blocked, rBSC1 mRNA decreased within 6 h by 38 +/- 11% in control but only by 15 +/- 15% in acid medium (P < 0.02), which represented an increase in the BSC1 mRNA half-life from approximately 7 to approximately 17 h. Furthermore, in a mouse TAL cell line, acid incubation for 16 h significantly increased (P < 0.02) the amount of BSC1 mRNA in cells transfected with the full-length mBSC1 cDNA but not in cells transfected with a mBSC1 cDNA lacking the 3'-UTR. These results demonstrate that acid pH enhances the stability of BSC1 mRNA probably by activating pathways that act on the AU-rich 3'-UTR of BSC1 mRNA, which contributes to the renal response to metabolic acidosis.

Regulation by glucocorticoids and osmolality of expression of ROMK (Kir 1.1), the apical K channel of thick ascending limb.

Mechanisms of regulation of ROMK channel mRNA and protein expression in medullary thick ascending limb (MTAL) were assessed in rat MTAL fragments incubated for 7 h. ROMK mRNA was quantified by quantitative RT-PCR and ROMK protein by immunoblotting analysis of crude membranes. Medium hyperosmolality (450 mosmol/kgH(2)O; NaCl plus urea added to isoosmotic medium) increased ROMK mRNA (P < 0.04) and protein (P < 0.006), and 10 nM dexamethasone also increased ROMK mRNA (P < 0.02). Hyperosmolality and dexamethasone had no additive effects on ROMK mRNA. NaCl alone, but not urea or mannitol, reproduced the hyperosmolality effect on ROMK mRNA. 1-Deamino-(8-d-arginine) vasopressin (1 nM) or 0.5 mM 8-bromo-cAMP had no effect per se on ROMK mRNA and protein. However, 8-bromo-cAMP abolished the stimulatory effect of dexamethasone on ROMK mRNA in the isoosmotic but not in the hyperosmotic medium (P < 0.004). In in vivo studies, the abundance of ROMK protein and mRNA increased in adrenalectomized (ADX) rats infused with dexamethasone compared with ADX rats (P < 0.02). These results establish glucocorticoids and medium NaCl concentration as direct regulators of MTAL ROMK mRNA and protein expression, which may be modulated by cAMP-dependent factors.