**6. APs as Urinary Biomarkers of Renal Injury**

Serum creatinine and blood urea nitrogen (BUN) are widely used markers of renal disease, but their sensitivity and specificity are limited, and they are not useful in distinguishing the stages of acute kidney injury (AKI) [90]. They lack sensitivity because they increase only when the renal lesion is evident. Thus, serum creatinine levels rise gradually, and the kidneys have already lost half of their functionality by the time normal levels are doubled [91,92]. Besides, normal levels of these markers can be affected by protein-rich diets, intestinal bleeding, muscle disease, and dehydration, generating false positives in the diagnosis of renal disease. There is a need for biomarkers to allow an earlier diagnosis of AKI, a better prediction of renal disease severity, and an improved assessment of adverse effects in drug development [90].

Various urinary biomarkers for the early detection of AKI have emerged over recent years [91,92], including tubular enzymes that are increased in urine after damage to the tubular epithelium [92], which can precede or even trigger renal dysfunction. Major advantages of urinary markers include the non-invasiveness of the sampling and their usefulness to elucidate the size and localization of tubular cell lesions and to detect the presence of necrosis or other alterations that evoke renal dysfunction [93]. The measurement of urinary enzymes and other urinary biomarkers may therefore be a valuable tool to obtain an early diagnosis during initial stages of renal disease and to follow its progression or regression, facilitating prediction of the prognosis. Urinary APs are considered promising and useful biomarkers of renal disease of different pathophysiological origin, and an automated photometric assay has been developed for their measurement [94].

APA, APN, and CAP are present in the brush border membrane of renal tubular cells [95], have molecular weights above 140 kDa, and are highly organ-specific. These conditions ensure the tubular origin of these urinary enzymes, which cannot pass easily into the urine through the glomerular barrier (Figure 4).

**Figure 4.** Main aminopeptidases studied as biomarkers of acute and chronic kidney injury.

APs participate in AngII metabolism, forming part of the renal renin–angiotensin–aldosterone system (RAAS) [96], which is elevated in renal diseases. One of these enzymes, alanine aminopeptidase (APN), is a brush border enzyme that was proposed in the early 1970s as a urinary marker of renal disease [97]. Later, Marchewka et al. [98] demonstrated that the measurement of APN and its isoforms was of diagnostic relevance in nephrolithiasis. They also observed significantly higher APN excretion in patients with glomerulonephritis than in controls and a positive correlation between urinary protein concentrations and APN activity [99]. Their findings supported the association between proteinuria and elevated activity of renal tubular brush border enzymes reported in other studies of chronic glomerulonephritis [100]. As an explanation of this association, these authors [99] proposed that the protein present in the ultrafiltrate produces a release of APN from the external membrane of renal tubule microvilli. Because of its external localization, its release into the urine can be caused by a weak destructive action and is not necessarily linked to disruption of the integrity of kidney tubule cells [101].

Jung et al. [102] reported that the urinary excretion of APN, alkaline phosphatase, γ-glutamyltransferase, and N-acetyl-β-D-glucosaminidase is age-dependent in humans. These enzymes were determined in random morning urine samples from 442 individuals aged from 5 days to 58 years, and their creatinine-normalized activity significantly decreased with increasing age. APN activity has also been proposed as a biomarker of the nephrotoxicity induced by vancomycin [101] or amphotericin B [103] in experimental models and in several human diseases, such as glomerulopathy [104], IgA nephropathy [105], and diabetes [106]. However, conflicting results have been published on its usefulness as a biomarker of renal function in kidney transplantation patients [107,108].
