Hydrochlorothiazide and acute urinary acidification: The "voltage hypothesis" of ENaC-dependent H+ secretion refuted.

AIM: The "voltage hypothesis" of H+ secretion states that urinary acidification following increased Na+ delivery to the collecting duct (CD) is ENaC dependent leading to transepithelial voltage-dependent increase in H+ secretion. We recently showed that furosemide acidifies the urine independently of ENaC activity. If the voltage hypothesis holds, hydrochlorothiazide (HCT) must acidify the urine. We here tested the acute effect of HCT on urine pH under normal and high ENaC expression. METHODS: Mice subjected to a control or a low-Na+ diet were anesthetized and infused (0.5 mL h-1 ) with saline. Catheterization of the urinary bladder allowed real-time measurement of diuresis and urine pH. Mice received either HCT (1 mg mL-1 ) or vehicle. Urinary Na+ and K+ excretions were determined by flame photometry. ENaC expression levels were measured by semi-quantitative Western blotting. RESULTS: (1) HCT increased diuresis and natriuresis in both diet groups. (2) K+ excretion rates increased after HCT administration from 18.6 ± 1.3 to 31.7 ± 2.5 µmol h-1 in the control diet group and from 23.0 ± 1.3 to 48.7 ± 3.0 µmol h-1 in the low-Na+ diet group. (3) Mice fed a low-Na+ diet showed a marked upregulation of ENaC. (4) Importantly, no acute changes in urine pH were observed after the administration of HCT in either group. CONCLUSION: Acute administration of HCT has no effect on urine pH. Similarly, substantial functional and molecular upregulation of ENaC did not cause HCT to acutely change urine pH. Thus, an increased Na+ load to the CD does not alter urine pH. This supports our previous finding and likely falsifies the voltage hypothesis of H+ secretion.

P2X receptors trigger intracellular alkalization in isolated perfused mouse medullary thick ascending limb.

AIMS: Extracellular ATP is an important regulator of renal tubular transport. Recently, we found that basolateral ATP markedly inhibits Na(+) and Cl(-) absorption in mouse medullary thick ascending limb (mTAL) via a P2X receptor. The underlying mechanism that mediates this ATP-dependent transport inhibition in mTAL is, however, unclear. The renal outer medullary K(+) channel (ROMK) is sensitive to intracellular pH where a reduction leads to closing of ROMK. We speculated that P2X receptor stimulation in the TAL could lead to changes in pHi , leading to a reduction in NaCl transport. METHODS: To test this hypothesis, we measured pHi in single perfused mouse mTALs using the fluorescent ratiometric dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethylester. RESULTS: Interestingly, basolateral ATP (100 µm) caused a prominent, reversible intracellular alkalization of mTAL, with an average pHi increase of 0.14 ± 0.02 (n = 14). This was completely abolished by the P2X receptor antagonist periodate-oxidized ATP (50 µm). The P2X receptor-mediated intracellular alkalization required the activity of the apical Na(+) /H(+) exchanger (NHE3). Typically, Gq -coupled receptors cause a significant acidification of tubular epithelial cells, which was confirmed in this study, by P2Y2 and Ca(2+) sensing receptor stimulation. CONCLUSION: This study reports that stimulation of basolateral P2X receptors causes a substantial intracellular alkalization in the isolated perfused mouse mTAL. This intracellular alkalization is mediated through an increased apical NHE3 activity, similar to what we previously observed when tubular transport is inhibited with furosemide. This increased NHE3 activity causes H(+) secretion in the mTAL and provides further support that the TAL is a site of urinary acidification.