The ugly duckling of urinary acidification: what is the contribution of the thick ascending limb of the loop of Henle to urinary acidification?

THE KIDNEY EXCRETES ACID in the form of titratable acidity and ammonium as well as in the form of free protons. The latter causes urinary acidification together with the reabsorption of filtered bicarbonate. Inborn or acquired forms of renal tubular acidosis (RTA) reduce the capacity of the kidneys to form ammonium and excrete acids (9). According to the functional defect and predominant site of renal damage, different subtypes of RTA have been classified: type I or distal RTA (dRTA), describing defects localized in the connecting tubule and collecting duct; type II or proximal RTA, caused by defects in the proximal tubule, and type IV or hyperkalemic RTA, summarizing different causes of relative aldosterone insufficiency or resistance in the distal nephron. Some authors use also the term type III RTA or combined RTA, referring to a mixed proximal and distal nephron pathology. Clinically, the distinction of these RTA subtypes depends on the detection of normal anion gap metabolic acidosis for all subtypes combined with bicarbonaturia or other signs of proximal tubule dysfunction (Fanconi syndrome) for type II proximal RTA or inappropriately alkaline urine in the face of metabolic acidosis for type I dRTA and the presence of hyperkalemia for type IV RTA. However, this distinction may not always be as obvious as it seems as compensatory mechanisms may mask overt metabolic acidosis or only partial defects may be present. Thus, specific provocation tests challenging the kidneys’ capability to reabsorb bicarbonate or to produce maximally acidic urine are routinely used to unmask such defects. To date, two types of urinary acidification tests are in clinical use: the ammonium chloride loading test and the furosemide-fludrocortisone (F F) test. The ammonium chloride loading test introduced by O. Wrong in the 1950s provides a metabolic acid load where hepatic metabolism of ammonium releases protons to be excreted by the kidney (21). A failure of the kidney to acidify urine below pH 5.3 (or, in some centers, 5.5) is diagnostic for type I dRTA. This test, although very reliable and serving as the “gold standard,” is very unpleasant for patients as ammonium chloride is poorly palatable and may cause nausea and vomiting. Alternatively, the F F test may be used, combining a mineralocorticoid such as fludrocortisone with a loop diuretic such as furosemide. This test was first introduced as a single application of furosemide by D. Batlle (1) aiming to provide pathophysiological insights and distinction into different causes of type I dRTA. It was further refined and validated by Walsh and colleagues (19) by adding a mineralocorticoid (e.g., fludrocortisone). The assumption underlying this test is that electrogenic Na absorption via the epithelial Na channel (ENaC) in principal cells in the connecting tubule and collecting duct causes a lumen negative potential that would enhance proton secretion by neighboring type A intercalated cells acidifying urine. The application of a mineralocorticoid increases ENaC activity, and the loop diuretic causes a shift in Na reabsorption from the thick ascending limb of the loop of Henle (TAL) to the connecting tubule and collecting duct, the major site of ENaC expression. This test is well tolerated by patients and appears to be also similarly reliable as the ammonium chloride loading test. However, new data presented in the American Journal of Physiology-Renal Physiology by de Bruijn and colleagues (5) challenge the interpretation of this test and ask the question of whether the TAL plays a much more important and hitherto underestimated role in urinary acidification. de Bruijn et al. report that furosemide not only blocks Na -K -2Cl cotransporter 2 (NKCC2) in TAL cells but also stimulates Na /H exchanger (NHE)3. The reduced absorption of Na via NKCC2 increases the Na gradient, providing a stronger driving force for NHE3 activity. Consequently, higher NHE3 activity would excrete more protons and acidify urine. These observations made in microperfusion experiments were further translated to the whole animal situation in technically very refined clearance experiments in mice by measuring in real-time furosemide-induced urinary acidification in the absence and presence of a specific NHE3 inhibitor. As expected, in the absence of the NHE3 inhibitor, furosemide caused a fall in urinary pH and an increase in the excretion of ammonium, whereas in the presence of the NHE3 inhibitor, urinary acidification and ammonium excretion were much reduced. Based on these data, the authors suggest that the TAL contributes to urinary acidification and that abnormal urinary acidification in response to furosemide may not only reflect a pathology in the connecting tubule and collecting duct (5). The TAL contributes to renal acid excretion by at least two processes: filtered bicarbonate, not reabsorbed by the proximal tubule, is mostly reabsorbed by the TAL involving NHE3 and V-type H -APases localized in the luminal membrane and a Cl /HCO3 exchanger together with a K -HCO3 cotransporter at the basolateral membrane (2, 4). Second, the TAL reabsorbs a major fraction of ammonium secreted by the proximal tubule into urine and accumulates ammonium in the medullary interstitium. This process involves NKCC2 accepting NH4 instead of K and basolateral NHE4 and Na -HCO3 cotransporter 1 involved in the basolateral release of ammonium (3, 11, 15, 18). Seminal work by Ullrich and Eigler (17) and Gottschalk et al. (8) in the late 1950s and early 1960s examined the sites of urinary acidification using micropuncture in rats and hamsters. They demonstrated that urine passing along the proximal Address for reprint requests and other correspondence: C. A. Wagner, Institute of Physiology, Univ. of Zurich, Winterthurerstrase 190, Zurich CH-8057, Switzerland (e-mail: [email protected]). Am J Physiol Renal Physiol 309: F431–F433, 2015; doi:10.1152/ajprenal.00296.2015. Editorial Focus