Abstract
Aims/hypothesisThe α subunit of the amiloride-sensitive epithelial sodium channel (α ENaC) is critical for the expression of functional channels. In humans and rats, non functional alternatively spliced forms of α ENaC have been proposed to act as negative regulatory components for ENaC. The purpose of this study was to examine the presence and consequently investigate the mRNA expression levels of alternatively spliced forms of α ENaC in kidney cortex of Dahl salt-resistant rats (R) versus Dahl salt-sensitive rats (S) on high salt and normal diets.MethodsUsing quantitative RT-PCR strategy, we examined the mRNA expression levels of previously reported α ENaC-a and -b alternatively spliced forms in kidney cortex of Dahl S and R rats on normal and four-week high salt diet and compared their corresponding abundance to wildtype α ENaC mRNA levels. We identified 2 novel non-coding C-terminus spliced forms and examined their mRNA expression in Dahl R versus S rat kidney cortex. We also tested the presence of five previously reported lung-specific α ENaC spliced forms in Dahl rat kidney cortex (CK479583, CK475461, CK364785, CK475819, and CB690980).ResultsPreviously reported α ENaC-a and -b alternatively spliced forms are present in Dahl rat kidney cortex and are significantly higher in Dahl R versus S rats (P < 0.05). Four-week high salt diet significantly increases α ENaC-b (P < 0.05), but not α ENaC-a transcript abundance in Dahl R, but not S rats. Two non-coding α ENaC spliced forms -c and -d are newly identified in the present study, whose levels are comparable in Dahl R and S rats. Compared to α ENaC-wt, α ENaC-a, -c and -d are low abundance transcripts (4 +/- 2, 110 +/- 20, and 10 +/- 2 fold less respectively), in contrast to α ENaC-b abundance that exceeds α ENaC-wt by 32 +/- 3 fold. We could not identify any of the five previously reported lung-specific α ENaC spliced forms (CK479583, CK475461, CK364785, CK475819, and CB690980) in Dahl rat kidney cortex.Conclusion/interpretationα ENaC alternative splicing might regulate α ENaC by the formation of coding RNA species (α ENaC-a and -b) and non-coding RNA species (α ENaC-c and -d). α ENaC-a and -b mRNA levels are significantly higher in Dahl R versus S rats. Additionally, α ENaC-b is a salt-sensitive transcript whose levels are significantly higher 4-weeks post high salt diet compared to normal salt diet in Dahl R rats. Among the four α ENaC transcripts (-a, -b, -c and -d), α ENaC-b is a predominant transcript that exceeds α ENaC-wt abundance by ~32 fold. α ENaC-a and -b spliced forms, particularly, α ENaC-b, might potentially act as dominant negative proteins for ENaC activity, thereby rescuing Dahl R rats from developing salt-sensitive hypertension on high salt diet. On the other hand, non-coding α ENaC-c and -d might assist alternative splicing, facilitate RNA processing, or regulate α ENaC as well as each other.
Highlights
The amiloride-sensitive epithelial sodium channel (ENaC) plays a key role in active sodium reabsorption by epithelia throughout the body, including kidney tubules, the lung, the distal colon, sweat and salivary glands, and the brain [1]
The inhibitor of apoptosis protein family was shown to be expressed at mRNA levels that were 2– 3% of the levels of the full-length transcript, yet it encodes a protein that accumulates 50-fold higher levels than fulllength and this accumulated protein competes with fulllength form for activity [18]
Presence of ENaC-a, -b, -c and -d in Dahl rat kidney cortex on normal and high salt diet Amplification of α ENaC-a, -b, -c and -d was done in Dahl S and R rat kidney cortex on normal and high salt diet (Figure 1A, B, C, D)
Summary
The amiloride-sensitive epithelial sodium channel (ENaC) plays a key role in active sodium reabsorption by epithelia throughout the body, including kidney tubules, the lung, the distal colon, sweat and salivary glands, and the brain [1]. ENaC activity is twice in kidneys of Dahl S versus R rats, possibly explaining the salt-sensitivity seen in Dahl S on high salt diet [9,10]. An example of the dominant negative role played by alternatively spliced forms on full length forms can be seen in the inhibitor of apoptosis protein family. The above facts have prompted us to examine the existence and the mRNA expression levels of α ENaC alternatively spliced forms -a and -b in Dahl R versus S rats on normal and 4 week-high salt diet, and to test the presence of five previously reported lung-specific α ENaC spliced forms in
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