Shotgun Proteomics of Isolated Urinary Extracellular Vesicles for Investigating Respiratory Impedance in Healthy Preschoolers.

Urine proteomic applications in children suggested their potential in discriminating between healthy subjects from those with respiratory diseases. The aim of the current study was to combine protein fractionation, by urinary extracellular vesicle isolation, and proteomics analysis in order to establish whether different patterns of respiratory impedance in healthy preschoolers can be characterized from a protein fingerprint. Twenty-one 3-5-yr-old healthy children, representative of 66 recruited subjects, were selected: 12 late preterm (LP) and 9 full-term (T) born. Children underwent measurement of respiratory impedance through Forced Oscillation Technique (FOT) and no significant differences between LP and T were found. Unbiased clustering, based on proteomic signatures, stratified three groups of children (A, B, C) with significantly different patterns of respiratory impedance, which was slightly worse in group A than in groups B and C. Six proteins (Tripeptidyl peptidase I (TPP1), Cubilin (CUBN), SerpinA4, SerpinF1, Thy-1 membrane glycoprotein (THY1) and Angiopoietin-related protein 2 (ANGPTL2)) were identified in order to type the membership of subjects to the three groups. The differential levels of the six proteins in groups A, B and C suggest that proteomic-based profiles of urinary fractionated exosomes could represent a link between respiratory impedance and underlying biological profiles in healthy preschool children.

Aminopeptidase activities as prospective urinary biomarkers for bladder cancer.

PURPOSE: Proteases have been implicated in cancer progression and invasiveness. We have investigated the activities, as opposed to simple protein levels, of selected aminopeptidases in urine specimens to serve as potential novel biomarkers for urothelial cancer. EXPERIMENTAL DESIGN: The unique urinary proteomes of males and females were profiled to establish the presence of a gender-independent set of aminopeptidases. Samples were also collected from patients with urothelial cancer and matched controls. A SOP for urine processing was developed taking into account hydration variation. Five specific aminopeptidase activity assays, using fluorophoric substrates, were optimized for evaluation of marker potential. RESULTS: Nineteen exopeptidases and 21 other proteases were identified in urine and the top-five most abundant aminopeptidases, identical in both genders, selected for functional studies. Depending on the enzyme, activities were consistently lower (p ≤ 0.05), higher or unchanged in the cancer samples as compared to controls. Two selected aminopeptidase activities used as a binary classifier resulted in a ROC curve with an AUC = 0.898. CONCLUSION AND CLINICAL RELEVANCE: We have developed functional assays that characterize aminopeptidase activities in urine specimens with adequate technical and intraindividual reproducibility. With further testing, it could yield a reliable biomarker test for bladder cancer detection or prognostication.

Urinary aminopeptidase activities as early and predictive biomarkers of renal dysfunction in cisplatin-treated rats.

This study analyzes the fluorimetric determination of alanyl- (Ala), glutamyl- (Glu), leucyl-cystinyl- (Cys) and aspartyl-aminopeptidase (AspAp) urinary enzymatic activities as early and predictive biomarkers of renal dysfunction in cisplatin-treated rats. Male Wistar rats (n = 8 each group) received a single subcutaneous injection of either saline or cisplatin 3.5 or 7 mg/kg, and urine samples were taken at 0, 1, 2, 3 and 14 days after treatment. In urine samples we determined Ala, Glu, Cys and AspAp activities, proteinuria, N-acetyl-ß-D-glucosaminidase (NAG), albumin, and neutrophil gelatinase-associated lipocalin (NGAL). Plasma creatinine, creatinine clearance and renal morphological variables were measured at the end of the experiment. CysAp, NAG and albumin were increased 48 hours after treatment in the cisplatin 3.5 mg/kg treated group. At 24 hours, all urinary aminopeptidase activities and albuminuria were significantly increased in the cisplatin 7 mg/kg treated group. Aminopeptidase urinary activities correlated (p<0.011; r(2)>0.259) with plasma creatinine, creatinine clearance and/or kidney weight/body weight ratio at the end of the experiment and they could be considered as predictive biomarkers of renal injury severity. ROC-AUC analysis was made to study their sensitivity and specificity to distinguish between treated and untreated rats at day 1. All aminopeptidase activities showed an AUC>0.633. We conclude that Ala, Cys, Glu and AspAp enzymatic activities are early and predictive urinary biomarkers of the renal dysfunction induced by cisplatin. These determinations can be very useful in the prognostic and diagnostic of renal dysfunction in preclinical research and clinical practice.

Salt sensitivity in experimental thyroid disorders in rats.

This study assessed salt sensitivity, analyzing the effects of an increased saline intake on hemodynamic, morphological, and oxidative stress and renal variables in experimental thyroid disorders. Six groups of male Wistar rats were used: control, hypothyroid, hyperthyroid, and the same groups treated with salt (8% via food intake). Body weight, blood pressure (BP), and heart rate (HR) were recorded weekly for 6 wk. Finally, BP and HR were recorded directly, and morphological, metabolic, plasma, and renal variables were measured. High-salt intake increased BP in thyroxine-treated rats but not in control or hypothyroid rats. High-salt intake increased cardiac mass in all groups, with a greater increase in hyperthyroid rats. Urinary isoprostanes and H(2)O(2) were higher in hyperthyroid rats and were augmented by high-salt intake in all groups, especially in hyperthyroid rats. High-salt intake reduced plasma thyroid hormone levels in hyperthyroid rats. Proteinuria was increased in hyperthyroid rats and aggravated by high-salt intake. Urinary levels of aminopeptidases (glutamyl-, alanyl-, aspartyl-, and cystinylaminopeptidase) were increased in hyperthyroid rats. All aminopeptidases were increased by salt intake in hyperthyroid rats but not in hypothyroid rats. In summary, hyperthyroid rats have enhanced salt sensitivity, and high-salt intake produces increased BP, cardiac hypertrophy, oxidative stress, and signs of renal injury. In contrast, hypothyroid rats are resistant to salt-induced BP elevation and renal injury signs. Urinary aminopeptidases are suitable biomarkers of renal injury.

Aminopeptidase activities, oxidative stress and renal function in Crotalus durissus terrificus envenomation in mice.

Acute renal failure is a serious condition of Crotalus bites, which could be treated with statins. The effects of Crotalus durissus terrificus venom (vCdt) and simvastatin on renal function, oxidative stress and representative plasma, urinary and renal aminopeptidase (AP) activities were evaluated in mice. Eighty percent LD50 of vCdt caused hyperuricemia and urinary hypoosmolality (100%) and hypercreatinemia (60%). Plasma neutral, pyroglutamyl and dipeptidyl IV and renal soluble and membrane-bound APs were susceptible to vCdt. Cortical and medullar oxidative stress (GSSG/GSH ratio) was increased by vCdt. Simvastatin (3mg/kg body wt.) altered urinary creatinine and urea, membranal protein in cortex and medulla, plasma neutral and dipeptidyl IV APs and most of renal APs in nonenvenomed, and exacerbated hypercreatinemia, but mitigated uricosuria, renal oxidative stress and protein increase in envenomed. Hyperuricemia and urinary hypoosmolality are early signs of indirect myotoxicity of vCdt with diagnostic significance. In kidney, oxidative stress and alteration of protein content and AP activities suggest membrane destruction, enzyme release and protein loss, which may be due to direct tissue damage. Plasma AP activities might be nephrotoxicity markers of C. d. terrificus envenomation. The deleterious effects of simvastatin on creatinemia and APs constitute important restrictions to its use within the antivenom therapy.

Enzymuria and beta2-mikroglobulinuria in the assessment of the influence of proteinuria on the progression of glomerulopathies.

The important role of the tubulo-interstitial system for the progression of glomerulonephritis (GN), is the cause of a continuous search for the proper markers of kidney tubules damage, which can be applied in clinical diagnosis. In the present work the activity of N-acetyl-beta-D-glucosamidase (NAG), its isoenzyme NAG-B, alanylaminopeptidase (AAP), gamma-glutamyltransferase (GGT), concentration of beta2-microglobulin (beta2M) and daily protein excretion in the urine of 37 patients with morphologically different glomerulopathies were measured. The serum creatinine was also controlled. The obtained results suggest that activity of NAG in the patients with GN has an intermediate connection with proteinuria and could be a cause of the inflammatory process of the kidney, but the activity of AAP is directly dependent on urine protein concentration. Systemic analysis of both partial and multiple correlation coefficients of the examined indicators creates new, additional possibilities in the estimation of activity and progress of GN.

The variation in excretory urinary glycyl-prolyl dipeptidyl aminopeptidase in dogs.

We studied the excretory variation of urinary glycyl-prolyl dipeptidyl aminopeptidase (GP-DAP, EC 3.4.14.5) in dogs. Adult domestic mongrel dogs (seven males and nine females, 7.5 to 13 kg bodyweight) which were considered to be healthy by laboratory tests were used. Urine and blood samples were obtained every four hours. Urine volume was measured for each sample and urine GP-DAP activity, and creatinine levels were determined. Creatinine clearance, creatinine excretion, GP-DAP activity and GP-DAP index (GP-DAP/Cr ratio) did not show any significant variation between the time periods. However, urine volume and urinary GP-DAP excretion significantly increased from 8:00 am to 12:00 pm. The GP-DAP index in spot urine samples showed low correlation with 24 hour GP-DAP excretion. In addition, a sex difference was observed in GP-DAP excretion. In conclusion, urinary GP-DAP excretion showed a circadian variation and sex difference. Therefore, GP-DAP in spot urine is not of use for the detection of renal disorders, and the 24-hour excretion value of GP-DAP should be determined.

Evaluation of urinary enzymes in dogs with early renal disorder.

We investigated urinary N-acetyl-beta-D-glucosaminidase NAG (EC 3.2.1.30), gamma-glutamyl transpeptidase gamma-GTP (EC 2.3.2.2) and glycyl-prolyl dipeptidyl aminopeptidase GP-DAP (EC 3.4.14.5) in dogs with heartworm disease and renal failure. In the renal failure dogs, the NAG, gamma-GTP and GP-DAP index were significantly higher than those in the healthy dogs. In the heartworm disease dogs with normal chest X-rays (HW I), none of the enzyme values was significantly different from those of the healthy controls. In the dogs with heartworm disease showing abnormal heart shadows on their chest X-rays (HW II), enzyme values were significantly higher than those in the healthy dogs (P < 0.01) and the HW I dogs (P < 0.01). Thus, these urinary enzymes tests are available for the early detection of renal disorders.

[Urinary enzymes in liver cirrhosis: useful early markers of renal damage?]. / Enzimi urinari in corso di cirrosi epatica: utili markers precoci di danno renale?

Some urinary enzymes (NAG, AAP, lysozyme) considered to be sufficiently sensitive and reliable markers of renal damage were controlled in 20 patients with cirrhosis of the liver and in 20 healthy control subjects. The results, stated as mean +/- SD, showed a statistically very significant increase (p < 0.01) of NAG and lysozyme in cirrhotics. Furthermore, this increase could be at least in part related with the seriousness of clinical condition. On the basis of these results, we think the urinary dosage of NAG and lysozyme is, in the subjects with liver cirrhosis, a bloodless method to show an early renal damage.

Specific developmental profiles of lysosomal and brush border enzymuria in the human.

Various enzymatic urinary activities have been proposed to assess renal proximal tubule damage in children, including neonates. Nevertheless comprehensive knowledge on the developmental aspects of physiological enzymuria is limited, particularly with regard to lysosomal and brush border enzymuria. Urinary activities of two lysosomal enzymes, N-acetyl-beta-D-glucosaminidase (NAG) and beta-galactosidase (GAL), and of two brush border enzymes, alanine aminopeptidase (AAG) and gamma-glutamyltransferase (GGT) were comparatively investigated in normal prematures (n = 28), term neonates (n = 52), infants aged less than 2 years (n = 19) and children (n = 33), and compared to urinary excretion of beta 2-microglobulin (B2M). Enzymatic activities were assayed using either spectrophotometrical (NAG, AAP, GGT) fluorimetrical (GAL) or radioimmunological (B2M) methods, and were related to urinary creatinine excretion. Developmental profiles of both the studied lysosomal enzymes and of B2M were similarly characterized with significantly decreasing values from prematures (NAG 9.29 +/- 1.44, GAL 2.26 +/- 0.26 IU/mmol creatinine, indicated as mean +/- SEM) to term neonates (6,94 +/- 0.58 and 1.76 +/- 0.15 IU/mmol creatinine, respectively) and older infants and children. Lysosomal enzymatic urinary activities correlated linearly with a coefficient of r = 0.75, (p < 0.05), while correlations between each lysosomal enzymatic activity and B2M urinary excretion were weaker.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary enzymes as biomarkers of renal injury in experimental nephrotoxicity of immunosuppressive drugs.

Urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG) and of alanine-aminopeptidase (AAP) was studied after administration of cyclosporine A (CSA A), FK 506, or the corresponding vehicles to salt-depleted rats. On days 7, 14, and 28 after treatment for CSA and day 14 after treatment for FK 506, measurements of the urinary enzymes, serum creatinine (SCr), creatinine clearance (ClCr), and blinded renal histology were done. After 1 week on CSA there was a dramatic increase of 489% in the urinary excretion of AAP (162.6 IU/g Cr, CSA vs. 27.6 IU/g Cr control, p < .03), a significant decrease of 32% in ClCr, a significant increase of 41% in SCr, and mild proximal tubular atrophy and vacuolization. After 2 or 4 weeks of CSA treatment there were no more differences in the urinary AAP between CSA and control rats, but the urinary excretion of NAG was increased: 29.6 IU/g Cr, CSA vs. 20.9 IU/g Cr, control, p < .03 on day 14 and 26.9 IU/g Cr, CSA vs. 21.5 IU/g Cr, control, p < .008 on day 28. At the same time there was a progressive decline of the ClCr, a progressive increase in the SCr, and an increase in the severity of the histological lesion. After 14 days of treatment with FK 506 we observed a striking elevation in urinary AAP (62.6 IU/g Cr, FK 506 vs. 36.0 IU/g Cr, control, p < .01) consistent with a significant decrease in ClCr, a significant increase in SCr, and a moderate proximal tubular vacuolization and atrophy.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of renal function parameters in the assessment of cis-platin induced nephrotoxicity.

The early renal function parameters (RFP), i.e. urinary alanine aminopeptidase (AAP), beta-galactosidase (beta GAL), N-acetyl-beta-D-glucosaminidase (NAG), retinol-binding protein (RBP), albumin (ALB), total protein (TP) and the conventional RFP plasma creatinine were assessed in 8 patients before and during treatment with the nephrotoxic antitumor agent cis-platin. Plasma creatinine increased during treatment with cis-platin. In all patients, acute tubular damage was revealed by early RFP. Albumin and total protein excretion patterns suggested alterations in glomerular function. The cumulative change in RBP excretion was related to plasma creatinine concentrations following cis-platin administration. The present study demonstrates that urinary RBP is a valuable parameter for the early assessment of cis-platin-induced nephrotoxicity.

N-acetyl-beta-D-glucosaminidase (NAG) and alanine aminopeptidase (AAP) excretion after acute administration of acetaminophen, salsalate and aspirin in rats.

The present study was performed to compare renal injury induced by high acute doses of both the nonacetylated salicylate, salsalate (SSA) and the acetylated salicylate, aspirin (ASA). As a marker of renal injury, urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG) and alanine aminopeptidase (AAP) were measured after oral administration of 600 mg/kg of each drug. Other tests to detect renal injury were also performed. Serum salicylic levels after SSA or ASA were determined in a different experiment. Acetaminophen (APAP) was used as a standard of renal toxicity. Both drugs increase NAG and to a greater extent, AAP excretion. Proteinuria and polyuria appear after both drugs. Changes in urinary sodium and potassium were also shown. Our results support the view that both acetylated and non-acetylated salicylates induce renal injury at toxicological doses.

Excretion of urinary enzymes after extracorporeal shock wave lithotripsy: a critical reevaluation.

The excretion of the urinary enzymes alanine aminopeptidase, alkaline phosphatase, gamma-glutamyl-transferase and N-acetyl-beta-D-glucosaminidase, and the 99mtechnetium-diethylenetriaminepentaacetic acid isotope clearance were studied in 35 patients treated with extracorporeal shock wave lithotripsy (ESWL*). Enzyme measurements were made before and consecutively on days 1, 2 and 5, and at 3 months after treatment. A control group investigated at the same intervals was included. Posttreatment enzyme values were not significantly different from those before treatment except for alkaline phosphatase on day 1 after ESWL. Some individuals had short-term increases of enzymuria that were greater than biological variations in the control group. The transient changes of enzymuria after ESWL had no predictive value for kidney function, since no decreased renal function was observed in individual patients with high enzyme excretions after ESWL.

Urinary proteins and enzymes as early indicators of renal dysfunction in chronic exposure to cadmium.

We tested the diagnostic sensitivity of various urinary analytes for detecting cadmium-induced nephropathy at an early stage. We investigated 73 healthy persons (control group 1) and individuals exposed to cadmium, either environmentally (n = 36, risk group 2) or occupationally (n = 62, exposed group 3). All data were related to limits of the central 95% reference intervals of the control group. The serum creatinine and ribonuclease values, indicators of the glomerular filtration rate, were not different in the three groups. In the exposed persons (group 3), proximal tubular indicators (low-M(r) proteins lysozyme, ribonuclease, retinol-binding protein, and alpha 1-microglobulin) were more often increased than the glomerular indices (higher-M(r) proteins transferrin, IgG, and albumin). Both the low-M(r) proteins and tubular enzymes were differently altered in their excretion rates. Alanine aminopeptidase, alkaline phosphatase, and N-acetyl-beta-D-glucosaminidase increased even in the risk group 2. alpha 1-Microglobulin was increased in the exposed persons whose cadmium excretion was < 5 mumol/mol creatinine. The combined determination of alpha 1-microglobulin and N-acetyl-beta-D-glucosaminidase exceeded the corresponding upper reference limits in 30% of group 2 and 39% of group 3. We recommend screening for these two analytes to detect cadmium-induced renal dysfunction at an early stage.

Effect of polyaspartic acid on CdCl2-induced nephrotoxicity in the rat.

We produced an animal model of CdCl2 nephrotoxicity in rats, and treated them with polyaspartic acid (PAA) to prevent renal damage. Male Sprague-Dawley (SD) rats (190-200 g) were used to induce proximal renal tubular damage by daily injection of CdCl2 3.0 mg/1,000 g body wt for 2 wk. CdCl2-exposed SD rats exhibited significant increases in urine volume, urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG), alanine aminopeptidase (AAP), and fractional excretion of sodium (FENa) and a decrease in the percentage of tubular reabsorption of phosphate (%TRP). Of these indicators of proximal tubular function, AAP and %TRP are more sensitive than NAG or FENa. No glycosuria or aminoaciduria, however, were observed. PAA markedly improved these indicators of proximal tubular function. Daily urinary protein excretion and creatinine clearance, on the other hand, did not change after administration of PAA. Cd concentrations in the cortex were 3 times higher than in the medulla, however, there were no differences between Cd-treated rats and PAA-treated rats. Our animal model is an excellent one for determining the effect of cadmium on renal proximal tubule damage. PAA appears to be useful in the treatment of CdCl2 nephrotoxicity.

Enzymuria in non-insulin-dependent diabetic patients: signs of tubular cell dysfunction.

1. To evaluate tubular damage in diabetic patients, we measured the 24 h urinary excretion of five enzymes (N-acetyl-beta-D-glucosaminidase, gamma-glutamyl transpeptidase, dipeptidyl aminopeptidase IV, alanine aminopeptidase and alkaline phosphatase) that originate in renal proximal tubular cells. 2. Studies were performed on 118 non-insulin-dependent diabetic patients, 59 non-diabetic patients with chronic renal disease and 47 normal control subjects. First, the correlation between renal function, glycaemic control and urinary enzyme excretion was investigated. Secondly, the subjects were treated by controlled diet therapy to assess the effects of better glycaemic control on urinary enzyme excretion. 3. Regardless of a diabetic or non-diabetic cause of renal dysfunction, all of the five enzymes showed abnormal urinary excretion in patients with renal insufficiency (serum creatinine concentration > 2.0 mg/dl). In diabetic patients, however, an increase in N-acetyl-beta-D-glucosaminidase excretion and a decrease in gamma-glutamyl transpeptidase excretion were noted even in those who had no signs of renal dysfunction, including microalbuminuria. Moreover, the excretion of these two enzymes had a higher degree of correlation with glycaemic control and renal function than did that of the other three enzymes. Multiple regression analysis revealed that excretion of N-acetyl-beta-D-glucosaminidase is best correlated with urinary protein (r2 = 0.35), whereas excretion of gamma-glutamyl transpeptidase is closely associated with glomerular filtration rate (r2 = 0.33). 4. In diabetic patients, diet therapy improved glycaemic control but had no effects on renal function, microalbumin excretion and beta 2-microglobulin excretion.(ABSTRACT TRUNCATED AT 250 WORDS)

[Comparative study on urinary parameters reflecting renal damage during chemotherapy for urological cancer].

We analyzed the excretion of the three urinary enzymes, N-acetyl-beta-D-glucosaminidase (NAG), alanine aminopeptidase (AAP), and glycyl-prolyl dipeptidyl aminopeptidase (GP-DAP) and the three urinary proteins, beta-microglobulin (beta 2-M), alpha-microglobulin (alpha 1-M), and albumin during the intravenous administration of anti cancer drugs in 4 prostatic cancer patients and 16 urothelial cancer patients. The patients with prostatic cancer were treated with VIP (vincristine, ifosfamide, peplomycin) chemotherapy and the patients with urothelial cancer were treated with MP (methotrexate, cisplatinum) or MEP (methotrexate, Etoposide, cisplatinum) combination chemotherapy. beta 2-M was the best parameter for drug-induced renal damage because of its sharp and large reactivities. Any urinary enzyme or protein especially albumin which had showed a markedly high value before the chemotherapy was not suitable as a urinary drug-induced renal damage parameter. There was a close resemblance in reactivity pattern between GP-DAP and AAP probably because of the same localization of these enzymes in the renal tubular bruch borders. In the patients with renal damage by chemotherapy, all parameters were changed showing two peaks or persisted with high values.

Urinary excretion of three specific renal tubular enzymes in patients treated with nonsteroidal anti-inflammatory drugs (NSAID).

Ten patients, mean age 51.50 +/- 3.03 years, with degenerative rheumatism on NSAID treatment without any sign of renal disease, and 11 control subjects, mean age 43.50 +/- 1.51 years, were studied. NSAID treatment was of 11.30 +/- 5.60 weeks duration in average, with ibuprofen, naproxen, or indomethacin. Urinary excretion of three specific renal tubular enzymes--AAP: alanine-amino-peptidase, GGT: gamma-glutamyl-transpeptidase, and beta-NAG: beta-N-acetyl-glucosaminidase, were determined in 8-h overnight urine samples, as well as GFR creatinine clearance/1.73 m2, urinary volume/8 h, specific gravity of the urine, proteinuria and glucosuria. In the group treated with NSAIDs, urinary excretion of the enzymes was significantly higher than in the control group--AAP: 1414.20 +/- 317.60, 864.20 +/- 94.42, p < 0.00001; GGT: 8034.6 +/- 1378.55, 5095.64 +/- 614.40, p < 0.00001, and beta-NAG: 1644.60 +/- 299.97, 964.82 +/- 142.00, p < 0.00001. Patients on NSAID treatment showed abnormal urinary excretion of AAP in 7/10 cases, of GGT in 6/10, and of beta-NAG in 7/10 cases. Duration of the treatment did not correlate with the urinary excretion of the enzymes. Age was in correlation with the urinary excretion of the enzymes only in the control group, r = 0.52, p < 0.005 for AAP, r = -0.43, p < 0.02 for GGT, and r = -0.23, p < 0.05 for beta-NAG.(ABSTRACT TRUNCATED AT 250 WORDS)