The main indication for evaluating urinary acidification in children is during the assessment and diagnosis of normal anion gap (hyperchloremic) metabolic acidosis, which can result, among other causes, from the loss of HCO₃⁻ from the gastrointestinal tract or kidney (proximal renal tubular acidosis –pRTA–), the addition of hydrochloric acid (HCl) or substances metabolized to HCl, or an impaired net renal acid excretion, as happens in distal renal tubular acidosis –dRTA– or in chronic kidney disease (CKD), when glomerular filtration rate (GFR) is significantly reduced (usually <20–25 ml/min/1.73 m²) (40).

The extrarenal (digestive) causes of hyperchloremic metabolic acidosis are the most common. In this context, when kidney function is preserved, UpH should be low and UNH₄⁺ should rise. The increase in UNH₄⁺ excretion is modest initially, but is maximized after 3–5 days if the stimulus persists (1, 3, 6). When more frequent extrarenal causes and CKD are excluded, an assessment of urinary acidification is indicated (40, 41). In these situations, an inappropriately high UpH with low UNH₄⁺, coexisting with metabolic acidosis, indicate an impaired distal urinary acidification (42). If distal acidification is preserved, efforts should be directed to evaluate renal HCO₃⁻ wasting (40). Usually, it is not necessary to measure UpH and UNH₄⁺ in most cases, since the underlying disorder is usually identifiable with a comprehensive history, physical examination and basic laboratory tests. However, the determination of UpH and UNH₄⁺ in single spot urine can be a useful and simple tool in the assessment of normal anion gap metabolic acidosis, especially when the cause is not clear or an underlying kidney pathology is suspected (40, 41).

Another less frequent indication for UpH and UNH₄⁺ measurement in pediatrics is during the realization of functional urinary acidification tests for the confirmatory diagnosis of dRTA, such as the oral NH₄Cl load (43) and the furosemide + fludrocortisone test (29, 44, 45). In healthy subjects, UpH should decrease below 5.3 in both tests (although this precise value is disputed) and UNH₄⁺ should rise up to 57 ± 14 (mean ± SD) µEq/min/1.73 m² in infants aged 1–16 months and 80 ± 12 µEq/min/1.73 m² in children aged 7–12 years (45) in the oral NH₄Cl load.

However, dRTA in children is mostly primary, caused by genetic alterations (41, 42, 46, 47) and the increasing availability of genetic testing has partly relegated functional tests in this context.

Incomplete dRTA is a disorder defined by an inability to maximally acidify urine in the absence of spontaneous metabolic acidosis (47). It is a condition that has been reported mostly in adults, since its first description in 1959 (48). In these patients, the acidification defect is milder and NH₄⁺ excretion is greater than in those with complete dRTA (49), a fact that may account for the absence of overt metabolic acidosis. The underlying mechanism that causes the impairment in urinary acidification is not well understood and may be dependent on the associated disorders (47).

Incomplete dRTA has been associated to recurrent calcium kidney stones in adults (50), nephrocalcinosis (50, 51), as well as to osteopenia or osteoporosis (52–54). In these cases, alkali therapy has shown to reduce stone formation and increase bone mass (54, 55). It has also been reported in sickle-cell disease (56), interstitial nephropathies (57) and autoimmune diseases [especially, Sjögren disease (58)].

The diagnosis of incomplete dRTA can be difficult to perform, since, by definition, serum acid-base balance parameters (including HCO₃⁻) are normal. Incomplete dRTA can be suspected in case of a persistently high UpH, but its confirmation requires the measurement of UpH and UNH₄⁺ in the same functional tests used to identify complete dRTA (42, 46, 47, 50). However, a low spontaneous UpH is usually considered to rule out an acidification defect without the need for functional tests (41, 47). Although, classically, the threshold value is 5.3 (48), cut-off points between 5.25 and 6.10 have been proposed and used. A list of publications on incomplete dRTA screening, along with the threshold values is provided in Table 1.

Incomplete dRTA in children has scarcely been reported and its manifestations are not well established. Nevertheless, and, unlike in adults, it has been associated to congenital abnormalities in the kidney and urinary tract (68, 69), asymptomatic hypocitraturia (59), active nutritional rickets (70) and it also has been reported in heterozygous carriers of gene mutations responsible for primary dRTA (71) who did not present spontaneous metabolic acidosis. Furthermore, whether it is also a cause of growth retardation in patients with vesicoureteral reflux and posterior ureteral valves (68, 69) without other growth impairment causes, remains an open question. Interestingly, sustained bicarbonate therapy has resulted in growth improvement in some of these cases (72). Therefore, incomplete dRTA evaluation could have important implications in clinical practice for diagnosing causes of growth failure in children, when the cause cannot be identified with routine testing (47, 73).

In addition to the very limited number of published studies on incomplete dRTA in pediatric age, data on UpH values in children are scarce and mostly limited to timed urine samples (74–76). Skinner et al. analyzed in 1996 the first morning UpH in 322 healthy children and found that only one child out of eight had a UpH ≤ 5.4, the median value being 6.0 (77). These results suggest that the utility of fasting UpH in detecting acidification defects is limited, at least when considered in isolation, and point out to the need for more data on pediatric reference UpH values and their relationship with other associated urinary acidification parameters, such as osmolality, electrolytes and, especially, UNH₄⁺.

Recent studies have emphasized the importance of measuring UNH₄⁺ in CKD. An impaired net acid excretion (and, accordingly, low NH₄⁺ secretion), with the consequent acid accumulation, contributes both to kidney injury (78) and to the pathogenesis of metabolic acidosis, which is a risk factor for progression of renal function deterioration and mortality (79, 80). Indeed, a low 24-hour UNH₄⁺ level (<20 mmol/day) has been found to be a marker of poor outcome in adult CKD patients without overt metabolic acidosis (80).

Acid accumulation is a well-known risk factor for progression of renal failure and mortality in CKD (80, 81), even when it is insufficient to cause clinically apparent metabolic acidosis, the so-called “eubicarbonatemic” acidosis (78). Acid accumulation in CKD depends, among other factors, on the reduction of GFR, the dietary acid and the integrity of distal acidification mechanisms. Acid-related kidney injury is not limited to patients with reduced GFR and is favored by high-acid diets, such as rich in protein Western diets (78). The identification of eubicarbonatemic acidosis, in order to prescribe dietary interventions or alkali therapy, even in early CKD stages, could decrease acid accumulation and slow CKD progress in these patients (78). Although acid accumulation can be estimated by serum HCO₃⁻ levels after an oral HCO₃⁻ load (82), more practical and easier surrogate parameters are needed. In this context, reduced urinary NH₄⁺ excretion might indicate risk for acid accumulation (78).

These data from adult CKD patients and the clinical implications of eubicarbonatemic acidosis need to be evaluated in children. Congenital kidney and urinary tract abnormalities, which are usually associated to tubulointerstitial injury, constitute a frequent cause of CKD in pediatric patients, especially in younger children (83). In these cases, where tubular function may be impaired already in early CKD stages, there might be a higher proportion of eubicarbonatemic acidosis than in adults, even when GFR is normal. This possibility and the potential clinical utility of UNH₄⁺ quantification in pediatric CKD patients, need to be explored.

The identification of a urinary acidification disorder needs a high degree of suspicion and can be difficult, especially when there is not a coexisting metabolic acidosis and serum HCO₃⁻ is within range, as in incomplete dRTA or eubicarbonatemic acidosis in CKD. Thus, there might be an important number of undiagnosed cases of acidification defects, both in adults and children, which can benefit from early identification and treatment. Since a confirmatory diagnosis requires a functional test, an accurate screening method is necessary. Ideally, this test should be non-invasive and easy to perform, such as the collection of a spot urine sample.

UpH is easy to measure but it cannot be used alone as a screening tool. UNH₄⁺ represents the most important part of net acid excretion and can provide more information on urinary acidification. Although traditionally not measured in clinical practice due to historical technical difficulties, which have been largely overcome, direct UNH₄⁺ quantification has gained in importance in the past few years in different conditions.

In fact, UpH and UNH₄⁺ measurement has shown to be feasible in clinical laboratories nowadays and its applications in clinical practice are starting to be discovered. The assessment of UpH and UNH₄⁺ should become a part of the evaluation of metabolic acidosis as a simple but informative diagnostic tool. Furthermore, evidence in adult patients also points out to their utility in cases of eubicarbonatemic acidosis and in order to identify incomplete dRTA.

However, there are very few data on UpH and UNH₄⁺ in pediatric population, so further research is needed to establish reference values in children, either in fasting conditions or in acidosis of nonrenal origin, and to explore the clinical applications of these measurements in metabolic acidosis, in CKD with a reduction of GFR and in incomplete dRTA associated disorders, including growth retardation of unknown cause. The recent availability of morning spot UNH₄⁺/creatinine ratio reference values in children (38) may clear the path for future studies.