Epithelial sodium channel (ENaC) subunit mRNA and protein expression in rats with puromycin aminonucleoside-induced nephrotic syndrome

Abstract

In experimental nephrotic syndrome, urinary sodium excretion is decreased during the early phase of the disease. The molecular mechanism(s) leading to salt retention has not been completely elucidated. The rate-limiting constituent of collecting duct sodium transport is the epithelial sodium channel (ENaC). We examined the abundance of ENaC subunit mRNAs and proteins in puromycin aminonucleoside (PAN)-induced nephrotic syndrome. The time courses of urinary sodium excretion, plasma aldosterone concentration and proteinuria were studied in male Sprague-Dawley rats treated with a single dose of either PAN or vehicle. The relative amounts of alphaENaC, betaENaC and gammaENaC mRNAs were determined in kidneys from these rats by real-time quantitative TaqMan PCR, and the amounts of proteins by Western blot. The kinetics of urinary sodium excretion and the appearance of proteinuria were comparable with those reported previously. Sodium retention occurred on days 2, 3 and 6 after PAN injection. A significant up-regulation of alphaENaC and betaENaC mRNA abundance on days 1 and 2 preceded sodium retention on days 2 and 3. Conversely, down-regulation of alphaENaC, betaENaC and gammaENaC mRNA expression on day 3 occurred in the presence of high aldosterone concentrations, and was followed by a return of sodium excretion to control values. The amounts of alphaENaC, betaENaC and gammaENaC proteins were not increased during PAN-induced sodium retention. In conclusion, ENaC mRNA expression, especially alphaENaC, is increased in the very early phase of the experimental model of PAN-induced nephrotic syndrome in rats, but appears to escape from the regulation by aldosterone after day 3.

Increased expression and apical targeting of renal ENaC subunits in puromycin aminonucleoside-induced nephrotic syndrome in rats

“…Thus this is likely to represent a key molecular basis for the sodium retention associated with PAN-induced nephrotic syndrome combined with the previously demonstrated increase in collecting duct Na-K-ATPase activity and protein abundance (13, 14). Thus our studies extend a previous study demonstrating that the total kidney abundance of ENaC subunits was not downregulated but maintained (3). Moreover, the present results extend this study by demonstrating the segmental specific upregulation of all three ENaC subunits, and most importantly, provide evidence for a significantly increased apical targeting by immunoelectron microscopy.…”

“…Thus this study directly points to a role of increased sodium reabsorption in the collecting duct and, hence, it may be hypothesized that dysregulation of the key sodium channels and transporters in the collecting duct may be responsible for this. A few studies subsequently demonstrated that sodium retention in PAN nephrotic rats is correlated with increased Na-K-ATPase activity and expression in the cortical collecting duct (CCD) (13,14), and a recent study demonstrated no major changes in the protein abundance of the epithelial sodium channel (ENaC) subunits in whole kidney (3).…”

Hyperaldosteronemia and Activation of the Epithelial Sodium Channel Are Not Required for Sodium Retention in Puromycin-Induced Nephrosis

“…Whether membrane recruitment of ENaC is associated with de novo synthesis of channel units remains difficult to address in vivo, in the absence of protein metabolic labeling. At variance with a previous report (11), our results show an increased level of ␤-ENaC mRNA in CCD from PAN-nephrotic rats. At the protein level, there are also discrepancies in the literature as Audigé et al (11) reported increased amounts of ␣-ENaC and no change in ␤-and ␥-ENaC in the kidney cortex of PAN-nephrotic rats, whereas Kim et al (3) described no change in ␣-and ␤-ENaC abundance and an increase of ␥-ENaC.…”

“…At variance with a previous report (11), our results show an increased level of ␤-ENaC mRNA in CCD from PAN-nephrotic rats. At the protein level, there are also discrepancies in the literature as Audigé et al (11) reported increased amounts of ␣-ENaC and no change in ␤-and ␥-ENaC in the kidney cortex of PAN-nephrotic rats, whereas Kim et al (3) described no change in ␣-and ␤-ENaC abundance and an increase of ␥-ENaC. From these data, it can be concluded that recruitment of pre-existing ENaC is the main cause of increased ENaC activity in PAN nephrosis.…”

“…In CCD, aldosterone increases both ENaC and Na ϩ ,K ϩ -ATPase activities through a biphasic mode: In the short term, it increases the targeting of intracellular pools of ENaC and Na ϩ ,K ϩ -ATPase to the apical and basolateral membrane, respectively (6,7), and in a later phase, it increases the cellular amount of ENaC and Na ϩ ,K ϩ -ATPase (8,9). Because plasma aldosterone level is increased in PAN-nephrotic rats at the time of sodium retention (3,10,11), it is usually assumed that hyperaldosteronemia mediates sodium retention in PAN nephrosis. Therefore, we investigated whether activation of ENaC in the CCD during PAN nephrosis is secondary to hyperaldosteronemia.…”