*Acute Kidney Injury*

AKI is defined by an abrupt increase in serum creatinine during the 48 h period after an insult responsible for functional or structural changes in the kidney, and it is mainly caused by the acute apoptosis or necrosis of renal tubular cells [109]. The early detection of AKI is a current preclinical and clinical research priority. The administration of nephrotoxic drugs that alter tubular function is the most common experimental model to relate the excretion of different biomarkers to renal dysfunction. Cisplatin is an antineoplastic drug with a potent nephrotoxic effect, mainly due to its action on the proximal tubule, causing AKI in experimental animal models and patients [110,111]. Its administration induces tubular and glomerular disturbances that lead to the development of interstitial fibrosis [112].

Our group investigated whether urinary activities of APN, APA, and CAP could serve as biomarkers of renal dysfunction by using a rat model of cisplatin-induced nephrotoxicity [113]. Their activity was determined in urine samples collected at 24, 48, and 72 h after cisplatin injection. Their urinary activity at 24 h post-injection was correlated with two renal function indicators (plasma creatinine and creatinine clearance) and their activity at two weeks post-injection was correlated with two indicators of structural damage (renal hypertrophy and interstitial fibrosis). A comparative analysis was also performed with other proposed urinary biomarkers of kidney injury, i.e., albumin, proteinuria, NAG, and NGAL. The area under the curve (AUC) was calculated to quantify the sensitivity and specificity of each marker to distinguish cisplatin-treated from control rats at 24 h post-injection (Figure 5). APN, APA, CAP, and DNPEP had an AUC value greater than 0.5, indicating that these enzymes are early biomarkers of kidney injury in cisplatin-treated rats. The greatest specificity and sensitivity values were observed for APN, which was the best variable to discriminate between treated and untreated animals, whereas an AUC value below 0.5 was observed for NAG and NGAL at this time point. A significant correlation was also found between AP activities at day 1 post-injection and functional and structural variables at day 14. It can therefore be concluded that the urinary activities of these APs may serve as early and predictive biomarkers of cisplatin-induced renal dysfunction. The measurement of urinary AP activities can be useful in preclinical research for the early detection and follow-up of renal damage and for the evaluation of drug nephrotoxicity, and it may offer a useful prognostic and diagnostic tool for renal diseases in the clinical setting.

**Figure 5.** ROC curves showing specificity and sensitivity for APN (AlaAp) (**A**), APA (GluAp) (**B**), proteinuria (**C**), albuminuria (**D**), N-acetyl-β-D-glucosaminidase (NAG) (**E**), and neutrophil gelatinase-associated lipocalin (NGAL) (**F**) to differentiate cisplatin-treated rats from control rats 24 h after injection of saline, 3.5 or 7 mg/kg of cisplatin (*n* = 8 each group). All cisplatin-treated rats displayed tubular dysplasia and interstitial fibrosis 14 days after injection. Urinary markers were expressed in daily total activity or excretion per 100 g of body weight. AUC = area under the curve. SENS 95 % = calculated sensitivity at 95 % of specificity.

#### **7. Quantification of Urinary APs**

APs activities can be readily quantified in biological samples by kinetic methods using photometric [94] or fluorometric [113] assays. Widely applied immunological techniques such as immunoblotting or ELISA can be used to analyze the amount/expression of these proteins in biological samples. Our group used ELISA and Western blot to measure the amount of APN excreted in the urine of cisplatin-treated rats and correlated its excretion 24 h post-injection with increased serum creatinine and decreased body weight at two weeks [114]. In addition, the quantification of APN by ELISA showed a very high correlation with urinary APA activity. The above findings confirm that the immunological determination of this enzyme can also serve as an early marker of renal damage. Further studies are warranted to compare the sensitivity and specificity of kinetic and fluorometric methods in other experimental models and in human diseases that involve renal dysfunction.

#### *7.1. Aminopeptidases in Urinary Vesicles and Exosomes*

Extracellular vesicles (EVs) released by the renal epithelium into the tubular fluid are excreted and may be detected in urine samples [115,116]; they include microvesicles and exosomes. Exosomes are nanovesicles (30–120 nm) released by epithelial cells throughout the urinary tract [117,118]. Thus, the exosomic fraction of urine contains potential biomarkers of various renal diseases. Since exosomes secrete mRNAs, miRNAs, and proteins that affect the function of recipient cells [119], it has been proposed that the biological role of exosomes in the kidney is to regulate the co-functionality of different sections of the nephron.

However, the content of biomarkers in urinary microvesicles has not been fully elucidated. The release of microvesicles into the urine has been studied in some renal diseases [120], and their content can also transmit biological signals within the kidney [121–124].

Our group therefore investigated whether the microvesicular and exosomic content of APA in urine was related to cisplatin-induced renal dysfunction in rats [125]. An increased APA content was observed in microvesicular and exosomic fractions at the peak of toxicity, 3 days post-cisplatin injection, and it showed a significant predictive correlation with serum creatinine, regardless of the method of normalization used for APA values (Figure 6). It was concluded that APA content and activity in urinary microvesicles and exosomes may serve as renal dysfunction biomarkers in this nephrotoxicity model.

**Figure 6.** Correlations of urinary activities of APN (AlaAp) (**A**), APA (GluAp) (**B**), and proteinuria (**C**) with percentage of renal interstitial fibrosis in urine samples of Zucker lean and obese rats (*n* = 10 each group). Cr = creatinine (adapted from reference [126]).

Thus, the determination of GluAp in microvesicular and exosomic fractions can be useful to study proximal tubular lesions independently of the glomerular filtration rate. From a technical viewpoint, it is interesting to note that the microvesicular fraction can be obtained more rapidly than can the exosomic fraction. In addition, the analysis of these fractions can avoid interference due to urea content, ionic strength, or other components of urine, because the samples are centrifuged and dissolved in a buffer. According to these observations, examination of the content of the microvesicular fraction can also be a relevant diagnostic tool in nephropathies.

#### *7.2. AKI Induced by Surgical Procedures*

Patients undergoing a cardiopulmonary bypass (CPB) can develop AKI as a post-surgical complication. The diagnosis of AKI in these patients is based on an increase in serum creatinine (SCr) concentration over baseline values during the 48 h postoperative period; however, the slow progressive rise of SCr can delay the detection of renal dysfunction. Kim et al. [127] analyzed the serum and urinary enzymatic activity of vasopressinase and SCr in 31 patients at different time points after CPB surgery. Results showed maximum serum and urine activity of vasopressinase at intensive care unit (ICU) admission in patients developing AKI as a complication of CPB earlier than the peak of SCr activity, which is usually observed at 48 h post-surgery. The authors therefore proposed vasopressinase (cystil AP, CAP) activity as an early biomarker for AKI. Animal models of renal ischemia also showed that AKI was related to vasopressinase activity [128].

In a similar study of 103 patients, our group observed a significant increase in urinary APA activity at ICU admission in patients who developed persistent AKI in comparison to those who did not experience AKI or developed only transient AKI. Urinary APA activity showed a higher sensitivity than SCr at this time point, obtaining a higher total number of correctly diagnosed cases in comparison to the application of criteria published by the Acute Kidney Injury Network [129]. These observations also indicate that tubular injury increases the risk of post-surgery AKI.
