Effect of high-salt diet on mean arterial pressure, renal epithelial sodium channels and aquaporin subunits expression levels in Spontaneously Hypertensive Rats

An increase in blood pressure (BP) by a high-salt (HS) diet may involve the changes in the expression of epithelium sodium channels (ENaCs) and aquaporins (AQPs) in the kidney which affect the sodium- and water-handling mechanisms. In the present study, spontaneously hypertensive rats (SHRs) and Wistar Kyoto (WKY) rats were exposed to HS and regular-salt (RS) diets for 6 weeks and fluid intake was monitored. After 6 weeks, mean arterial pressure (MAP) and plasma hormonal activity of atrial natriuretic peptide (ANP), levels of angiotensin II (Ang II), aldosterone and arginine vasopressin (AVP) were determined. The expression of mRNA and protein levels of ENaC and AQP subunits in kidneys were quantified by real-time PCR and Western blotting. High-salt diet caused higher MAP only in SHRs and higher fluid intake in both strains of rats when compared with their respective controls on RS diet. The plasma levels of Ang II and aldosterone were low in both SHRs and WKY rats fed with HS diet. Meanwhile, plasma ANP activity was high in both strains of rats on HS diet; whilst the AVP showed vice versa effects. The renal expression of mRNA and protein levels of α- and γ-ENaCs was lowered by HS diet in both SHRs and WKY rats. Although β-ENaC mRNA and protein expression levels were depressed in SHRs but they were enhanced in WKY rats. On the other hand, AQP-1, 2 and 7 mRNA and protein expression levels were lowered in both strains of rats fed with HS diet, while that of AQP-3, 4 and 6 showed no significant changes. The suppression of mRNA and protein expression levels of ENaC and AQP subunits suggests that the HS-induced increase in the MAP of SHRs may not be due to the renal sodium and water retention solely.


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In all the reported studies, inappropriate sodium and water retention by ENaC 101 and AQP subunits have been shown to be involved in the pathogenesis of HPN. Most 102 of the studies on the effect of HS were performed in Dahl salt-sensitive and salt-103 resistance rats as well as SD rats. But studies on the ENaCs and AQPs dysregulations 104 in SHRs, the rat model that shares similar pathophysiology with essential HPN in 105 human population, as a consequence of HS diet were far from complete. Therefore, in 106 the present study, we used SHRs to investigate the expression level of both ENaC and 107 AQP subunits as a result of HS intake. We hypothesised that chronic HS diet intake 108 affects expressions of ENaC and AQP subunits in the kidney which lead to sodium and 109 water retention, respectively, and the subsequent increase in BP.

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Ethical approval 113 The study was carried out in the Department of Physiology and Medical Biotechnology

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ENaC was also downregulated in SHS when compared with SRS; however, the result 309 was not significant (Fig 4C). On the other hand, the mRNA expression level of β-ENaC 310 was found to be also lower in SHS when compared with SRS; however, the results were 311 not significant. Meanwhile, there was no significant change in the expression of β-

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ENaC in WHS when compared with WRS ( Fig 4B).   In this present study, we found that SHRs on a HS diet showed significantly higher

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MAP when compared with SHRs on a RS diet (Fig 1)

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We also found that both the rat strains fed with HS showed higher fluid intake 378 when compared to their respective controls (Fig 2). It is well known that the rise in 379 plasma sodium content will increase plasma osmolarity, which causes a rise in

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In the meantime, the present study showed higher plasma ANP activity in both 388 SHRs and WKY rats fed with HS diet when compared with their respective controls 389 ( Fig 3A). It is well acknowledged that ANP is synthesised by atria in heart and secreted 390 into the bloodstream in response to stretching of right atrial muscle cells by increased 412 the plasma aldosterone level in both SHRs and WKY rats was lower when compared 413 with their respective controls ( Fig 3C). It is well documented that the synthesis of 414 aldosterone increases in response to low plasma sodium so that sodium will be retained 415 in the cell [52]. As such, the increase in sodium as in the present study would certainly 416 secrete low aldosterone. Therefore, it is not surprising to see the low plasma aldosterone 417 level in both SHRs and WKY rats fed with HS diet. The higher plasma aldosterone 418 level in SHRs compared with WKY rats also explains the higher BP in SHRs as that of 419 WKY rats. Furthermore, the release of aldosterone is also dependent on the level of 420 Ang II; thus, the low Ang II might also lead to a low aldosterone level in both strains 421 of rat. Meanwhile, the plasma AVP level was found to be slightly higher in SHRs fed 423 with HS salt diet whilst lower in WKY rats ( Fig 3D)

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The effect of HS diet on the mRNA expression and protein distribution of ENaC 435 subunits were also investigated in the present study. Both the mRNA and protein 436 expression of α-, β-and γ-ENaC subunits were downregulated and lowered, 437 respectively, in SHRs fed with HS diet when compared with SHRs on RS diet (Fig 4).

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In the present study, the mRNA of AQP showed various expression patterns in 475 SHRs and WKY rats in response to HS diet. In SHS (SHRs being fed with HS diet), 476 the mRNA levels of AQP1, AQP2 and AQP7 were found to be lower when compared 477 to SRS (SHRs being fed with RS diet) (Fig 5). Consistent with the downregulation of 478 AQP1, AQP2 and AQP7, the protein levels of these AQPs were also depressed when 479 compared with their controls (Fig 7). Similar changes in the mRNA and protein 480 expressions of AQPs were demonstrated by WKY rats. Meanwhile, the mRNA 481 expression levels of AQP3 and AQP4 (Fig 5C and D) were found to be enhanced in 482 both strains of rats being fed with HS diet. However, the protein level of AQP4 (Fig   483 7D) in SHS was found to be lower when compared with its counterpart. Interestingly, 484 AQP6 displayed contra-expression in mRNA and protein level in SHRs and WKY rats; 485 in which mRNA level of SHR (SHS vs SRS) was enhanced but the protein level was 486 depressed whilst mRNA level in WKY rats (WHS vs WRS) was lower but protein level 487 was enhanced. All these observations in mRNA and protein levels of AQPs have led to 488 interesting point to discuss.

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Aquaporin1 is the major water channel in renal proximal tubule and loop of  516 AQP2 level in the present study could be due to the compensatory mechanism other 517 than via AVP; though plasma AVP was slightly higher (Fig 3D) in SHRs. Meanwhile, 518 the low mRNA and protein levels of AQP2 in WKY rats as result of HS diet may 519 directly due to the low AVP in these rats. Nevertheless, the observation in WKY rats in 520 the present study is in accordance with the study by Roxas et al. [93], which showed 521 that low expression of AQP2 transcript in SD rats fed with HS diet. In addition, stimulation of thirst by HS diet may also be a possible explanation for the suppressed 523 AQP2 in both strains of rats which excessive water drinking keeps circulating AVP 524 levels very low, resulting presumably in suppressed AQP2 levels in the kidneys [95].

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Both AQP3 and AQP4 are constitutively localised in basolateral membrane in 526 principal cells of collecting duct. To be more precise, AQP3 is found in cortical and 527 outer medullary collecting duct, whereas AQP4 is located primarily in inner medullary 528 collecting ducts. They both represent potential exit pathways i.e. the increased 529 intracellular water absorbed by AQP2 is transported to blood by AQP3 and AQP4 [35] 530 according to an osmotic gradient. In the present study, both these AQPs showed 531 upregulation in mRNA expression level in both strains of rats fed with HS diet ( Fig 5C   532 and D); whilst, SHRs showed lower protein expression of AQP4. The dramatic 533 upregulation of AQP3 and AQP4 mRNA expression as a consequence of HS diet 534 indicates that the increased water reabsorption in collecting duct may contribute to 535 extracellular volume expansion, which is a typical characteristic of SSH. This is further 536 supported by our findings (Fig 1) that showed the higher MAP in SHRs and WKY rats 537 consuming HS diet. Furthermore, SHRs are known to have a high AQP3 level [27,32].
538 The upregulation of AQP3 is in consistent with higher protein expression of AQP3 in 539 the present study ( Fig 7C). However, the downregulation of protein expression of 540 AQP4 in SHRs remains to be elucidated.

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On the other hand, AQP7 localised at the brush border of proximal straight 542 tubule where AQP1 is also located has been classified as aquaglyceroporins because of 543 its credibility to transports water and glycerol as well as urea just as AQP3. In the 544 present study, expressions of AQP7 at mRNA and protein level (Fig 5F and 7F