Na+/H+ exchanger regulation and hypertension in genetically hypertensive rats

The interest in sodium in the pathogenesis of essential hypertension has a long history. Increased sodium retention leading to hypertension can be demonstrated experimentally with animal models such as the deoxycorticosterone acetate-salt hypertensive rat. However, increased exchangeable sodium and/or plasma volume has never been established in human essential hypertension, and these are more often found to be low [1–3]. It is common for an initiating change in disease to be attenuated by compensating mechanisms but this cannot cause a reversal of the abnormality. Thus, it is very unlikely that sodium retention has a major role in human essential hypertension. The development of the ‘spontaneously’ or ‘genetically’ hypertensive rat model (SHR), which did not require surgical and/or pharmacological intervention to become hypertensive, allowed investigations of the mechanism of hypertension. These studies revealed that exchangeable sodium in the SHR was higher than the parent Wistar–Kyoto (WKY) strain [4,5]. Initially, this difference (as well as some others) suggested that the mechanism of hypertension in the SHR was different to that in human essential hypertension. However, an elegant study back-crossing SHR with WKY rats showed that the increased exchangeable sodium and increased blood pressure were independently inherited [5]. Again, this called into question the role of sodium retention in the pathogenesis of hypertension and this observation is relevant to the paper by Kobayashi et al. [6] in this issue of the journal. There is also a history of interest in Na+/H+ exchanger activity in essential hypertension. This came from observations on blood cells [7] where activity is due to NHE1 and is unlikely to reflect Na+/H+ exchange activity involved in renal tubular sodium reabsorption. However, it was suggested that increased NHE1 activity may have a role in cell proliferation and the structural changes in arteries and myocardium [8]. The study by Kobayashi et al. [6] specifically concerns NHE3 in renal proximal tubule (PCT) cells and is therefore relevant to renal sodium reabsorption. There are two issues associated with their paper: (i) can changes in NHE3 activity in PCT cells explain increased blood pressure in the SHR and (ii) can NHERF expression explain changes in NHE3 activity in PCT cells in the SHR. In the SHR, NHE activity was indeed approximately two-fold higher than in WKY rats at both 4 and 11 weeks. However, there was no significant difference in blood pressure between SHR and WKY rats at 4 weeks although it was markedly higher in SHR at 11 weeks. This can be reconciled if an increase from 2 to 4.5 units of Na+/H+ exchange activity causes no change in blood pressure but a change from 4.5–7 units causes a very large rise in blood pressure [6]. Alternatively, it shows that NHE3 activity does not comprise a large factor in the higher blood pressure found in SHR compared to WKY rats. The latter interpretation agrees with the early experiments showing that sodium retention and hypertension do not cosegregate in these rats [5]. It is possible that the PCT cells had changed in culture from their ‘hypertensive’ condition in vivo but the SHR cells did retain higher Na+/H+ exchanger activity. However, this may be an inherent genetic difference, which may or may not be causally related to their higher blood pressure. The comparison with Dahl rats raises further questions. Dahl salt-resistant (DR) rats on a high-salt diet (HS) had NHE activity as high as SHR at 11 weeks. However, whereas SHR were very hypertensive, DR had no change in blood pressure. The only change in NHE activity observed was lower activity in the Dahl salt-sensitive (DS) rats in response to high-salt diet (DS-HS), which could be explained by decreased NHE3 expression. If higher NHE3 activity causing increased sodium retention was involved in the development of hypertension, it would be expected to persist at a higher level in the DS-HS than in the DR-HS, rather than the reverse situation that was observed. The differences in NHE3 activity were apparently different from those described in a previous study [9] using brush border vesicles, which found no difference in activity between DS-LS and DS-HS. Possible changes to cultured cells from their condition in vivo is offered as an explanation, but the small differences in activity relative to variation in the measurements in the two studies also appears to be a possible cause for the apparent statistical disagreement. The changes in NHE activity were not those expected as a possible cause of hypertension in either study. There has been interest in the mechanism of increased sodium retention in the SHR irrespective of its role in the pathogenesis of hypertension. The Na+/H+ exchanger isoform 3 (NHE3) in the brush border of the proximal tubule cells has an important role in sodium reabsorption. Hence, increased NHE3 activity has been considered as a possible cause of increased sodium reabsorption. The paper by Kobayashi et al. [6] reports experiments investigating the role of regulatory factors, NHERF1 and 2, in relation to this phenomenon. These are inhibitory factors and, accordingly, the hypothesis is that a decrease in their expression would lead to increased NHE3 activity and hence increased sodium retention. Whereas NHE activity was different, NHE3 mRNA expression was the same in SHR and WKY rats and showed very little change between 4 and 11 weeks, although protein levels were not demonstrated. The latter may be important because it has been reported that NHE3 mRNA levels are normal despite increased NHE3 protein levels and activity in SHR [10]. In this case, consideration of NHERF1 and 2 as an explaination of the increased NHE3 activity is unnecessary. However, as predicted by the hypothesis, NHERF1 levels were significantly lower in the SHR with higher NHE activity than the WKY rats. Closer examination indicates that, with no increase in NHE3 mRNA expression between 4 and 11 weeks of age, a 60% increase in NHE activity occurred with a 15% increase in the inhibitory NHERF1 [6]. This casts doubt on the relevance of NHERF1 in determining NHE3 activity. However, NHERF1 is thought to be the most important factor in the regulation of NHE3 in PCT cells with NHERF2 present in the glomerulus and renal vasculature, and more important in regulation of the ROMK K+ channel [11]. For an inhibitory factor, NHERF2 shows the expected decrease for an increase in NHE activity with age but not the expected difference between SHR and WKY rats. If it is assumed that NHERF1 and 2 are equivalent inhibitors of NHE3 then it appears that the sum of these two factors changed very little with either age or SHR [6]. Although, during culture, PCT cells could possibly change from their condition in vivo, the Na+/H+ exchanger activity and NHERFs were measured in cells in the same condition and, accordingly, this explanation is not tenable for the lack of relationship between NHERF expression and Na+/H+ exchanger activity. One further technical reservation is the use of GAPDH as a ‘housekeeping’ gene for mRNA quantitation. The widespread assumption that the expression of GAPDH is constant may be unsafe [12]. NHERF1 and 2 were named following recognition of their first demonstrated activity, but they may also have a wider role in cell physiology with respect to the cytoskeletal interaction of proteins that span the lipid bilayer [13]. The insertion of transport proteins into the lipid bilayer to become active, and their subsequent withdrawal to cease activity (as occurs with NHE3 in response to blood pressure elevation) [14,15] is a control mechanism that is widely used by cells. NHERF1 and 2 appear to be of major importance in these trafficking processes [13] and these may not just involve NHE3. For example, NHERF2 has been shown to specifically interact with a calcium channel, and this may be important in the regulation of calcium signalling [16]. Intracellular calcium has complex effects on NHE3 by mediating the formation of NHE3–cell membrane complexes and also in stimulating PKCα translocation to the cell membrane where it associates with NHERF2. This complex with active PKC is essential for the NHERF2-mediated inhibition of NHE3 by down-regulation of the transporter from the cell membrane [17]. NHERF2 may also influence the activity of a phospholipase C isoform, and hence phosphoinositol signalling [18]. The function of NHERF1 and 2 in complexes at the cytoskeletal–lipid bilayer interface suggests that NHERF activity may be dependent on other proteins in those complexes. Both NHERF1 and 2 have ezrin as part of their membrane binding complex [11,13,19,20]. NHERF2 interacts with SGK1 in the regulation of NHE3 activity [21] and polymorphisms in the SGK1 gene have been associated with raised blood pressure [22]. In addition, NHERF2 and SGK1 also interact to alter the activity of the ROMK1 K+ channel [23]. This channel has increased activity in DS rats and has been suggested as the cause of sodium retention and hypertension [24]. There appears to be little reason to believe that NHE activity and NHE3 expression are causative factors in hypertension in either SHR or Dahl rats. There is also uncertainty regarding the importance of NHERF expression with respect to changes in NHE activity in SHR PCT cells. However, continued interest in the NHERFs may be justified by their effects being much wider than just those on NHE activity.