The Polarized Effect of Intracellular Calcium on the Renal Epithelial Sodium Channel Occurs as a Result of Subcellular Calcium Signaling Domains Maintained by Mitochondria.

The renal epithelial sodium channel (ENaC) provides regulated sodium transport in the distal nephron. The effects of intracellular calcium ([Ca(2+)]i) on this channel are only beginning to be elucidated. It appears from previous studies that the [Ca(2+)]i increases downstream of ATP administration may have a polarized effect on ENaC, where apical application of ATP and the subsequent [Ca(2+)]i increase have an inhibitory effect on the channel, whereas basolateral ATP and [Ca(2+)]i have a stimulatory effect. We asked whether this polarized effect of ATP is, in fact, reflective of a polarized effect of increased [Ca(2+)]i on ENaC and what underlying mechanism is responsible. We began by performing patch clamp experiments in which ENaC activity was measured during apical or basolateral application of ionomycin to increase [Ca(2+)]i near the apical or basolateral membrane, respectively. We found that ENaC does indeed respond to increased [Ca(2+)]i in a polarized fashion, with apical increases being inhibitory and basolateral increases stimulating channel activity. In other epithelial cell types, mitochondria sequester [Ca(2+)]i, creating [Ca(2+)]i signaling microdomains within the cell that are dependent on mitochondrial localization. We found that mitochondria localize in bands just beneath the apical and basolateral membranes in two different cortical collecting duct principal cell lines and in cortical collecting duct principal cells in mouse kidney tissue. We found that inhibiting mitochondrial [Ca(2+)]i uptake destroyed the polarized response of ENaC to [Ca(2+)]i. Overall, our data suggest that ENaC is regulated by [Ca(2+)]i in a polarized fashion and that this polarization is maintained by mitochondrial [Ca(2+)]i sequestration.

Basolateral P2X4channels stimulate ENaC activity in Xenopus cortical collecting duct A6 cells.

The polarized nature of epithelial cells allows for different responses to luminal or serosal stimuli. In kidney tubules, ATP is produced luminally in response to changes in luminal flow. Luminal increases in ATP have been previously shown to inhibit the renal epithelial Na⁺ channel (ENaC). On the other hand, ATP is increased basolaterally in renal epithelia in response to aldosterone. We tested the hypothesis that basolateral ATP can stimulate ENaC function through activation of the P2X4receptor/channel. Using single channel cell-attached patch-clamp techniques, we demonstrated the existence of a basolaterally expressed channel stimulated by the P2X4agonist 2-methylthio-ATP (meSATP) in Xenopus A6 cells, a renal collecting duct principal cell line. This channel had a similar reversal potential and conductance to that of P2X4channels. Cell surface biotinylation of the basolateral side of these cells confirmed the basolateral presence of the P2X4 receptor. Basolateral addition of meSATP enhanced the activity of ENaC in single channel patch-clamp experiments, an effect that was absent in cells transfected with a dominant negative P2X4receptor construct, indicating that activation of P2X4channels stimulates ENaC activity in these cells. The effect of meSATP on ENaC activity was reduced after chelation of basolateral Ca²âº with EGTA or inhibition of phosphatidylinositol 3-kinase with LY-294002. Overall, our results show that ENaC is stimulated by P2X4receptor activation and that the stimulation is dependent on increases in intracellular Ca²âº and phosphatidylinositol 3-kinase activation.

Distribution of inflammatory mediators in carotid and femoral plaques.

BACKGROUND: Although atherosclerosis is a known systemic process, carotid and femoral atherosclerotic plaques are associated with distinctive complications and therapeutic outcomes. The purpose of this study was to evaluate inflammatory markers associated with carotid and femoral plaques. STUDY DESIGN: Carotid and femoral endarterectomy specimens were harvested from surgical patients. The carotid specimens were further sectioned into central, peripheral, and relatively normal regions. Expressions of matrix metalloproteinase (MMP)-2 and MMP-9 in carotid and femoral specimens were compared and the distributions of MMP-2, MMP-9, and CD40 within the carotid specimens were further analyzed. Messenger RNA (mRNA) levels were measured with real-time polymerase chain reaction and proteins with ELISA assay and Western blot. RESULTS: Despite no significant difference in the MMP-2 mRNA levels between carotid and femoral specimens, the common femoral specimens had significantly lower MMP-2 protein production and higher MMP-9 mRNA and protein expressions than those of the carotid specimens (p = 0.015, p = 0.03, and p = 0.034, respectively). Among carotid specimens, MMP-2 mRNA level was significantly lower in the central region (p < 0.01) and consistent with the distribution pattern of MMP-2 proteins. Interestingly, despite significantly lower MMP-9 mRNA expression in the central region of carotid plaques, active MMP-9 protein level was significantly higher. CD40 mRNA and protein levels were also higher in the central region. Immunohistochemistry analyses showed substantially increased macrophages and CD40 in the central region of the carotid plaques. CONCLUSIONS: This study emphasizes that femoral and carotid plaques are distinct entities and that distribution of inflammatory molecular markers varied within these advanced atherosclerotic plaques. Additional analyses are warranted.