Increased Renal Sodium Absorption by Inhibition of Prostaglandin Synthesis During Fasting in Healthy Man. A Possible Role of the Epithelial Sodium Channels

Thomas G Lauridsen; Henrik Vase; Jørn Starklint; Carolina C Graffe; Jesper N Bech; Søren Nielsen; Erling B Pedersen

Disclosures

BMC Nephrology. 2010;11 

In This Article

Discussion

The present study of healthy subjects showed that ibuprofen decreased FENa and increased u-ENaCβ both during a 24 hours fasting period, during the first two hours after 24 hours fasting, and during the following period with hypertonic saline infusion. The increased tubular sodium absorption during ibuprofen treatment might be attributed to increased sodium transport from the tubular lumen via the epithelial sodium channels. Our hypothesis was not falsified regarding this point. However, ibuprofen decreased u-AQP2 and u-PGE2, and did not change p-AVP, u-c-AMP, urinary output, and free water clearance during any of the study periods. Thus, our results demonstrated that u-AQP2 was reduced, although an increased AQP2 trafficking might be expected, when the antagonizing effect of prostaglandins on the vasopressin mediated effect on the principal cells was reduced/eliminated during ibuprofen treatment. Consequently, our hypothesis that reduction of prostaglandin synthesis would increase u-AQP2 was falsified.

In the present study we have used a new and original method to evaluate sodium reabsorption in the principal cells in the distal tubules using a radioimmunoassay of the β-fraction of the protein in the epithelial sodium channel. The amount of ENaCβ in urine is supposed to reflect the activity of sodium transport via the epithelial sodium channels just as u-AQP2 reflects the functional status of the AQP2 water channels (31). Our analyses showed that the assay has a satisfactory reliability. In addition, we demonstrated a significant correlation between changes in urinary sodium excretion and changes in u-ENaCβ. Thus, our results are in accordance with u-ENaCβ being a biomarker of the transport of sodium via ENaC during acute studies, presumably reflecting up- and down regulation β-ENaC expression and sodium transport via ENaC. It is well known that ibuprofen reduces renal sodium excretion. Our study adds new information, since our data might suggest that prostaglandin inhibition possibly reduce renal sodium excretion via an increased transport through ENaC.

In our study, ibuprofen significantly reduced FENa during all parts of study to a level around 35% lower than placebo. The response in FENa during hypertonic saline infusion was the same during treatment with ibuprofen and placebo, and the difference, i.e. the lower level of FENa during ibuprofen treatment, persisted during the infusion. Several animal studies have shown that PGE2 has a direct inhibitory effect on sodium chloride transport in the collecting ducts.[2,5,13,14] In rats, this is regulated by the EP2 receptor.[15] We found that u-ENaCβ was significantly increased during ibuprofen treatment. Thus, the decrease in FENa might, at least partly, be explained by an increased transport of sodium from the tubular lumen via ENaC. However, we cannot exclude that the antinatriuretic effect of ibuprofen was also attributed to some extent by an upregulation of the Na-K-2Cl cotransporter in the ascending limb of Henle's loop.[16] U-ENaCβ was increased by ibuprofen without an increase in either p-AVP or u-c-AMP, which indicates that AVP does not seem to play a direct regulatory role in the increased u-ENaCβ. However, the marked reduction in u-PGE2 makes it likely that the reduced prostaglandin level results in the increase sodium transport via ENaC, and this explanation is supported by evidence from animal studies.[7,17–19]

Ibuprofen did not change the response in the effect variables qualitatively during hypertonic saline infusion, but renal sodium excretion was reduced as hypothesized. After hypertonic saline infusion u-ENaC was reduced significantly at 60 minutes, and at the same time p-ANP tended to increase. An increase in ANP might be responsible for reduction in u-ENaCβ during this condition, and could be seen as a homeostatic mechanism to prevent sodium and fluid expansion.[6] Aldosterone is an important regulator of the transport activity via ENaC. Aldosterone stimulates the MR receptor. The result is an increased transcription of genes, which code for proteins involved in sodium transport, i. e. ENaC and Na-K-ATPase.[3,20] In-vitro and in-vivo studies have shown that aldosterone increases the synthesis of the α-fraction of ENaC in the distal tubuli.[21]

In our study, we found that u-ENaCβ was reduced in the ibuprofen group during and after hypertonic saline infusion. We did not measure a reduction in p-aldosterone. This might be due to the fact that our study was too short to allow the regulatory effect of aldosterone.

A time delay in aldosterone secretion has previously been demonstrated in another study, in which an acute infusion of hypertonic saline infusion decreased PRA, but plasma aldosterone was unchanged.[22] We think the short observation time after the hypertonic saline infusion might explain the fact that p-aldosterone was unchanged in the present study.

During placebo treatment, hypertonic saline resulted in a significant increase in u-ENaCβ. This increase was associated with an increased FENa. The explanation of this phenomenon is not clear for the time being. A considerably decrease in the renal sodium absorption more proximally in the nephron might be compensated for by an increase in absorption in the distal part of the nephron, but additional studies are required to determine the precise use of u-ENaCβ as biomarker for ENaC activity during these conditions. However, a rise in the intracellular sodium concentration was suggested to be a trigger for the feedback inhibition of sodium absorption via ENaC in rats when expressed in xenopus laevis oocytes.[23] Thus, the reduction in u-ENaCβ during sodium loading could alternatively be attributed to this inhibitory feedback mechanism of increased intracellular sodium on sodium transport via ENaC.

In a previous study, we showed that fasting decreased u-AQP2 and reduced the stimulating effect of vasopressin on u-AQP2.[24] Fasting increased prostaglandin synthesis, and the refractoriness to vasopressin during fasting was proposed to be due to an antagonizing effect of prostaglandins on AQP2 trafficking. Thus, it would be reasonable to hypothesize that an increase in u-AQP2 could be expected by ibuprofen treatment. On the contrary, ibuprofen reduced u-PGE2 markedly in the present study during all conditions i.e. in 24 hours urine, in the period shortly after 24 hours of fasting, and during infusion of hypertonic saline, as expected. However, ibuprofen also reduced u-AQP2 during all parts of the study, and thereby falsified our hypothesis that reduction of prostaglandin synthesis would increase u-AQP2.

A previous study in humans showed that prostacyclin infusion increased u-AQP2 excretion, and this seems to be in agreement with our results.[25] Apparently, a discrepancy exists between animal studies on the one hand and studies in man on the other. In animals, prostaglandin decreased APQ2 trafficking,[2,26] and inhibition of prostaglandin synthesis by infusion of indomethacin increased urinary excretion of AQP2.[4] In healthy man prostaglandin and prostaglandin-inhibitors had the opposite effects, as demonstrated in the present study and by others [25]

We found that ibuprofen significantly reduced u-PGE2 due to a reduced renal prostaglandin synthesis. This is in accordance with other studies in healthy man.[27,28] Prostaglandin E2 increased urinary output and sodium excretion by inhibition of AVP stimulated water absorption, inhibition of sodium absorption and stimulation of basal water absorption.[8] Different subtypes of prostaglandin receptors mediate this effect from the basolateral part of the tubular cells.[29] Receptor stimulation by PGE2 reduced sodium reabsorption in the thick ascending limb of Henle and in the cortical collecting duct, and reduced AVP-induced water absorption in cortical collecting duct.[9,30,31] Both p-AVP and u-c-AMP were unchanged by ibuprofen, but simultaneously we measured a clear reduction in u-AQP2. Thus, the vasopressin-c-AMP axis was not involved in the reduction in u-AQP2. It is generally accepted that prostaglandin E2 antagonizes the effect of vasopressin on AQP2 trafficking via the EP3 receptor. Activation of this receptor inhibits adenylyl-cyclase, reduces the level of c-AMP, causing increased urinary output. However, the close relationship between c-AMP production and increased AQP2 trafficking was challenged in a recent experimental study.[32] Activation of the EP3 receptor inhibited AQP2 trafficking in inner medullary cells from rats, despite high levels of c-AMP, probably due to an cAMP- and Ca2+-independent Rho activation. Rho promotes the formation F-actin which hinders AQP2 coated vesicles reaching the apical membrane.[32] Our study in healthy man is in agreement with these findings, in the sense that we found a reduction in u-AQP2 excretion without changes in urinary excretion of c-AMP. This suggests that adenylyl cyclase activity did not contribute to our results regarding u-AQP2 excretion. The changes in p-AVP during hypertonic saline infusion was as expected, and the changes were not significantly different during ibuprofen treatment compared with placebo.

As expected, we measured a marked reduction in urinary sodium excretion during ibuprofen treatment as reflected in the decrease in FENa, but no significant changes in urinary output and free water clearance. However, some tubular reabsorption of water must take place simultaneously with sodium reabsorption due to the effect of ibuprofen on basal renal water transport. Most likely, this fact explains our findings, i.e. the lack of increase in urinary output and free water clearance despite decreased u-AQP2 during ibuprofen treatment. Both p-AVP and u-c-AMP were unchanged by ibuprofen, but simultaneously we measured a clear reduction in u-AQP2. Thus, the vasopressin-c-AMP axis was not involved in the reduction in u-AQP2. The changes in p-AVP during hypertonic saline infusion was as expected, and the changes were not significantly different during ibuprofen treatment compared with placebo.

However, other regulatory factors than vasopressin and prostaglandins may influences the expression of AQP2 and u-AQP2 such as the renin-angiotensin-aldosterone system, the natriuretic peptide system, and the sympathetic nervous system.[1,6,33–38] We measured higher levels of p-BNP and a clear tendency to an increase in p-ANP simultaneous with a tendency to a lower level of p-Ang II and p-Aldo during ibuprofen treatment. Most likely, these changes in hormones with both vasoactive- and sodium- and water regulating properties are secondary to the sodium retention induced by ibuprofen. This is supported by the fact that p-albumin fell significantly, and that body weight tended to increase, presumably due to an expansion of the extracellular fluid volume. Animal studies support this explanation. Angiotensin II stimulated/enhanced AQP2 expression,[36,38] and angiotensin II receptor blockade reduced AQP2 expression.[1] Aldosterone agonism and antagonism increased and decreased AQP2 expression, respectively.[34] We suggest that the tendency to reduced levels of the components in the renin-angiotensin-aldosterone system contributes to the reduced level of u-AQP2 during ibuprofen treatment in fasting healthy humans.

The role of ANP in the regulation of intracellular distribution of AQP2 was addressed in rats.[6] ANP-infusion had no immediate effect on the intracellular localization of AQP2, but after 90 minutes of ANP-infusion an increased apical targeting of AQP2 was noted. This was regarded as either a direct or compensatory effect to volume depletion to avoid dehydration. A human study with head out water immersion demonstrated increased AQP2 expression accompanied by an increase in p-ANP.[33] These findings do not prove any causal relationship, as several other homeostatic systems as the sympathetic nervous system and the renin-angiotensin-aldosterone system are also influenced by the intervention. Thus, the effect of the natriuretic peptide system on AQP2 trafficking and urinary excretion is not fully elucidated. Accordingly, we cannot rule out that the increased levels of the natriuretic peptides after ibuprofen treatment had modulated u-AQP2 in our study.

Inhibition of the renal prostaglandin synthesis might be dangerous in patients with heart failure, lever disease and renal insufficiency. It can result in sodium and water retention and hypertension. We did not measure any changes in blood pressure and the increase in body weight was marginal. Most like, this can be attributed to the fact that we studied healthy subjects.

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