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.

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.

Radiation injury in the human kidney: a prospective analysis using specific scintigraphic and biochemical endpoints.

Renal function was prospectively analyzed in 26 evaluable patients, irradiated to various doses on their kidneys for neoplastic disease. Glomerular function was assessed by 99mTc-DTPA renography, creatinine clearance, and serum beta 2-microglobulin, whereas tubular function was monitored by 99mTc-DMSA scintigraphy, urine beta 2-microglobulin, urine N-acetyl glucosaminidase, and alanine aminopeptidase and a urine concentration test. In the patients given the highest irradiation dose to the entire left kidney, that is, 40 Gy in 5 1/2 weeks, glomerular and tubular functional impairment, as assessed scintigraphically, progressed at a rate of 2.0 +/- 1.0% (+/- 1 SD) and 2.0 +/- 0.5% per month, respectively, down to 30-40% after 3 to 5 years. The overall glomerular function, as assessed by creatinine clearance, decreased by only 20%. In the patients irradiated unilaterally on the upper pole to 40 Gy in 4 weeks, glomerular and tubular function in the left kidney deteriorated at 0.75 +/- 0.33% and 0.75 +/- 0.20% per month in the first 2 years, down to 75-80% at 5 years. This smaller reduction was due to shielding of a part of the left kidney. No changes were observed, thus far, after bilateral whole kidney irradiation to 17-18 Gy in 3 1/2 weeks. The concentration capacity of the kidney after total volume irradiation was not impaired. There was a trend for an increase in diastolic blood pressure in 3 out of 5 patients given the high dose irradiation to the entire left kidney and in 2 out of 7 patients irradiated on the upper pole of the left kidney. The progressive nature of the radiation nephropathy stresses the need for long term follow-up to determine more accurately the "tolerance dose" of the human kidney for irradiation.

Renal toxicity during therapy with gentamicin or tobramycin.

Twenty-seven patients who had normal pretherapy renal clearance by the [125I]iothalamate test were randomly assigned either gentamicin or tobramycin for therapy of infections due to susceptible bacteria. No patients were critically ill or had evidence of bacteremia. Mean age and duration of therapy were 51 years and 14 days, respectively, for 15 patients treated with gentamicin, and 45 years and 13 days for 12 patients treated with tobramycin. At the completion of therapy, six (40%) gentamicin and seven (58%) tobramycin patients had a decrease in renal clearance of at least 14% below baseline. The mean decrease was 26% in the gentamicin group and 23% in the tobramycin group. Simultaneous increases in serum creatinine concentrations (greater than or equal to 0.2 mg/dl) occurred in only 4 (31%) of the 13 patients. Of four patients who had renal clearance studies repeated 3 weeks to 6 months after therapy, two had stable function, but at 16 to 19% below baseline. Mean urinary concentration of N-acetyl glucosaminidase and alanine aminopeptidase increased faster and to higher levels with gentamicin than with tobramycin. However, on an individual patient basis, they were not predictive of a decrease in renal clearance in either therapy group.

Monitoring serum aminoglycoside concentrations in children with amphotericin B nephrotoxicity.

We prospectively studied the effect of amphotericin B therapy on aminoglycoside clearance in 20 consecutive children during the remission-induction phase of chemotherapy for acute myelocytic leukemia. Increases (greater than 50%) in the half-life for aminoglycoside excretion were not associated with antileukemic or aminoglycoside therapy alone but occurred in 12 of 17 children when amphotericin B was added to the antimicrobial regimen. Seven children had impaired aminoglycoside clearance without increases (greater than 50%) in serum creatinine; hence the resulting adjustments in aminoglycoside dosage would not have been made had we relied solely on serial measurements of serum creatinine. Evidence for increased excretion of the renal enzymes N-acetyl-beta-D-glucosaminidase and alanine aminopeptidase during amphotericin B therapy suggested that damage to proximal tubular cells may contribute to the renal impairment that has been associated with this drug. Our findings underscore the value of monitoring serum aminoglycoside concentrations in children being treated with amphotericin B.

Enzymuria in neonates receiving continuous intravenous infusion of gentamicin.

Urinary excretion of the tubular enzymes NAG and AAP was investigated during gentamicin treatment of 105 newborn infants. The values found for NAG and AAP show a significant positive correlation. The urinary excretion of NAG was on the average 92% higher during gentamicin treatment as compared with non-treatment periods in the same newborn infant (33 infants). The same tendency applied to AAP. Newborn infants receiving continuous intravenous infusion of gentamicin were not found to be at greater risk of nephrotoxicity than those receiving intermittent gentamicin treatment, using NAG and AAP as an index of nephrotoxicity. The changes in NAg and AAP within treatment periods were studied. During gentamicin treatment an insignificant average increase in the urinary excretion of NAG occurred, whereas a significant decrease was found during non-treatment periods. A significant negative correlation was found between urinary excretion of NAG and birth weight/gestational age. The long-term effect of the higher excretion of NAG and AAP in newborn and adult patients during aminoglycoside treatment is unknown.

Comparison of N-acetyl-beta-D-glucosaminidase and alanine aminopeptidase activities for evaluation of microangiopathy in diabetes mellitus.

The activities of urinary N-acetyl-beta-D-glucosaminidase (NAG) and alanine aminopeptidase (AAP) were measured in 207 diabetic patients and 57 healthy controls, and the relationship of these enzymes to different stages of diabetic microangiopathy was studied. Diabetics with clinical proteinuria had higher urinary NAG and AAP (17.7 +/- 1.9 and 42.8 +/- 4.9 U/g creatinine, mean +/- SE, respectively) than healthy controls (1.8 +/- 0.1 and 10.0 +/- 0.4) or diabetics without proteinuria. Among diabetics without proteinuria, NAG excretion in those with retinopathy was slightly higher than in those without (6.4 +/- 0.5 v 5.4 +/- 0.4), and AAP in those with retinopathy was significantly higher than in those without (23.0 +/- 1.5 v 17.4 +/- 0.8, P less than 0.01). Urinary albumin measured by radioimmunoassay and lysozyme in diabetics with retinopathy but without proteinuria was higher than those without retinopathy (P less than 0.001 and P less than 0.01). The increase in albumin was the greatest in diabetics with long duration of the disease (greater than or equal to 8 years); however, NAG and AAP increased more significantly in those with high hemoglobin A1c than in patients with long duration.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal tubular enzyme effects of clarithromycin in comparison with gentamicin and placebo in volunteers.

This study assessed the potential nephrotoxicity of clarithromycin in comparison with gentamicin and placebo. Increased urinary excretion of alanine aminopeptidase (AAP) and N-acetyl-beta-D-glucosaminidase (NAG) served as markers of renal tubular injury. The study utilised a multiple-dose, double-blind, randomised, parallel group design. 14 healthy male subjects received 1 of 3 treatment regimens: (a) clarithromycin 500 mg orally every 12h for 13 doses and intravenous placebo every 8h (n = 5); (b) oral placebo every 12h and intravenous placebo every 8h (n = 4); and (c) intravenous gentamicin 1.7 mg/kg every 8h for 19 doses and oral placebo every 12h (n = 5). 24h urine collections were obtained daily for determinations of AAP and NAG activities. Gentamicin produced statistically significant increases (p less than 0.0001) in AAP and NAG excretion, with increases as early as the first and second day of dosing. Clarithromycin, when compared with placebo, did not produce significant elevations in AAP or NAG activity. On the basis of these data, it is unlikely that usual doses of clarithromycin have significant potential for causing nephrotoxicity.

Cumulative renal tubular damage associated with cisplatin nephrotoxicity.

We assessed the acute and chronic effect of multiple courses of cisplatin therapy on renal tubules by monitoring the urinary excretion of alanine aminopeptidase, N-acetyl-beta-D-glucosaminidase, and total protein. Urine specimens were obtained before and after doses of cisplatin (90 mg/m2) given to 12 patients. Each dose of cisplatin induced transient increases in enzyme excretion, followed by proteinuria 3-5 days later. Transient enzymuria after the last cisplatin dose was significantly greater than that after the first dose. Moreover, persistent increases in urinary N-acetyl-beta-D-glucosaminidase and serum creatinine concentrations over pretherapy levels indicated chronic renal tubular damage. Our findings disclosed striking differences between patients in susceptibility to progressive nephrotoxicity.

Assessment of renal function during high-dose cis-platinum therapy in patients with ovarian carcinoma.

Five courses of cis-dichlorodiammine platinum (II) (100 mg/m2) were given to 22 patients with advanced stage III and IV ovarian cancer. Renal function was assessed by measurement of creatinine clearance, urinary osmolality and urinary B2-microglobulin (B2MG) in all patients, and by urinary alanine aminopeptidase (AAP) and N-acetyl-B-glucosaminidase (NAG) excretion in seven patients. Serum creatinine, creatinine clearance, urinary osmolality, and urinary B2-microglobulin were within the reference ranges and did not change significantly after five courses of cis-platinum in any patient. There was a significant increase in the urinary excretion of both enzymes (AAP and NAG) within 2 days of cis-platinum administration (NAG P less than 0.05 and AAP P less than 0.07). There was evidence of a cumulative effect during treatment for AAP (P less than 0.025).

Tubular nephrotoxicity during long-term ifosfamide and mesna therapy.

The nephrotoxic effects of ifosfamide were assessed in 18 children and adolescents given cumulative doses of 32-112 g/m2 (1.6 g/m2 per day in sequential 5-day courses) with the uroprotectant mesna (1.2 g/m2 per day). Tubular nephrotoxicity was evaluated by measuring the urinary concentrations of N-acetyl-beta-D-glucosaminidase (NAG), alanine aminopeptidase (AAP), and total protein before and during sequential courses of therapy. Of 15 patients who had normal levels of tubular markers before ifosfamide therapy, only 1 developed a persistent increase in baseline values of the three tubular markers with the sixth course of ifosfamide. Although transient increases in the excretion of these markers were observed during each 5-day course of ifosfamide, the magnitude did not increase over sequential courses in these 15 patients. Of the remaining three patients who had increased NAG levels before ifosfamide therapy, two showed a progressive increase in enzymuria and proteinuria, and serum creatinine concentrations increased in a single patient who had obstructive uropathy. Our data suggest that children with normal renal function can be given large cumulative amounts of ifosfamide in fractionated doses with little risk of progressive clinical nephrotoxicity.

Carboplatin (CBDCA), iproplatin (CHIP), and high dose cisplatin in hypertonic saline evaluated for tubular nephrotoxicity.

We compared the acute tubular nephrotoxicity of three platinum compounds in children and adults with solid tumors by monitoring the urinary excretion of alanine aminopeptidase, N-acetyl-beta-D-glucosaminidase, and total protein. Cisplatin (100 mg/m2) was administered with mannitol, or at a twofold larger total dosage (50 mg/m2 per day for 4 days) in a 3% saline infusion. Carboplatin (300 mg/m2) was administered in combination with 5-fluorouracil, and iproplatin was administered in dosages ranging from 216 to 388 mg/m2. Enzymuria and proteinuria induced by cisplatin at a total dosage of 200 mg/m2 on a divided schedule did not significantly differ from that observed for the single 100 mg/m2 dose. Enzymuria and proteinuria induced by carboplatin and iproplatin were significantly less than that for cisplatin; however, one patient developed chronic tubular damage after three courses of carboplatin, and the acute tubular toxicity of iproplatin in one of 15 patients was exceptional. Our findings support the value of administering cisplatin in hypertonic saline on a divided schedule as a strategy to reduce acute tubular damage. Although carboplatin and iproplatin are less nephrotoxic than cisplatin, occasionally patients experience subclinical acute or chronic tubular damage that may lead to overt nephrotoxicity with continued therapy.

Assessment of renal toxicity by urinary enzymes in patients receiving chemotherapy with 8-methyl-8-acetylenic-putrescine.

Renal toxicity was assessed in 19 patients receiving methyl acetylenic putrescine (MAP), an irreversible inhibitor of ornithine decarboxylase. Patients received 250 mg t.d.s. for up to 13 weeks. This dose effectively inhibited the target enzyme, as shown by elevations in decarboxylated S-adenosyl methionine levels. No significant nephrotoxicity was observed in these patients as determined by plasma urea, creatinine and creatinine clearance measurements, although minor elevations of the urinary enzymes lactate dehydrogenase, N-acetyl-beta-glucosaminidase, alkaline phosphatase and alanine aminopeptidase were observed. As this could represent sub-clinical renal damage, caution should be exercised when using MAP in combination with other cytotoxic drugs.

Quality control material for activity determinations of urinary enzymes.

We examined the stability of N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30), alanine aminopeptidase (EC 3.4.11.2), alkaline phosphatase (EC 3.1.3.1), gamma-glutamyltransferase (EC 2.3.2.2), and lysozyme (EC 3.2.1.17) in urine prepared by gel filtration and supplemented with albumin, or ethylene glycol, or ethylene glycol plus albumin during storage at -20 degrees C for a period of 12 months. The stability was assessed by linear regression analysis of monthly values versus time. All enzymes except for gamma-glutamyltransferase could be considered stable for about one year in all three control materials provided that maximum change of 10% of the starting enzyme activity is accepted as tolerable. If ethylene glycol is used as stabilizer, its suitability must be tested and its inhibitory effect on enzyme activities must be taken into account in intermethod comparisons, because in some methods, it may be removed in a pretreatment step.

Diagnostic significance of urinary enzymes in detecting acute rejection crises in renal transplant recipients depending on expression of results illustrated through the example of alanine aminopeptidase.

In 82 renal transplant recipients we measured the excretion of urinary alanine aminopeptidase (EC 3.4.11.2) activity daily after transplantation until patients' discharge from hospital. Enzyme excretion was expressed as volume activity (U/L), time-related output (U/4h) and in relation to the excreted creatinine. Diagnostic sensitivity, specificity, efficiency and likelihood ratios were calculated from all three expressions with regard to diagnosis of acute rejection crises. The term "enzyme activity per creatinine excreted" has the highest diagnostic potential.

Variation of urinary enzymes N-acetyl-beta-glucosaminidase, alanine-aminopeptidase, and lysozyme in patients receiving radio-contrast agents.

A urinary enzyme pattern consisting of two lysosomal enzymes, N-acetyl-beta-glucosaminidase and lysozyme, and one enzyme originating from kidney tubular brush border membrane, alanine-aminopeptidase, were studied in 30 patients undergoing intravenous urography and arteriography. N-acetyl-beta-glucosaminidase and lysozyme showed the greatest diagnostic sensitivity and were still abnormal on the fifth day after the administration of radio contrast agent. The results, which are statistically significant (Student's t test), suggest that radio-contrast agents are potentially nephotoxic.

Urinary enzyme activities in patients treated with gold and other antirheumatic drugs.

We have studied 48 rheumatoid arthritis (RA) patients treated with nonsteroidal antiinflammatory drugs (NSAIDs), except salicylates, in 31 of whom parenteral gold was associated as therapeutic agent. In order to assess initial tubular involvement, the activities of some urinary enzymes were measured: N-acetylglucosaminidase (NAG, EC 3.2.1.30), microsomal amino-peptidase (MAP, EC 3.4.11.2) and gamma-glutamyltransferase (GGT; EC 2.3.2.2). Results were compared with a control group of 51 subjects of similar age, with no rheumatic symptoms and normal renal function. Both groups of patients (31 with gold therapy and 17 without) showed a significantly increased activity of NAG in urine, but the increase was greater in those treated with gold. MAP and GGT were not elevated significantly in either group. There was no correlation, however, between the increase of NAG and the cumulative dose of gold. NAG, MAP and GGT activities in serum yielded no relevant information. All the usual tests of renal function were also normal. Determination of NAG in urine may be regarded as a sensitive test, capable of detecting selective involvement of renal tubular cells, whose final diagnostic and prognostic significance merits further evaluation.

Identification of inhibitors of urinary alanine aminopeptidase.

Amino acids and ammonia were identified as natural inhibitors of urinary AAP. In urines from healthy volunteers approximately one half of the inhibition could be accounted for by amino acids and ammonia. At the measured concentrations, histidine, ammonia and phenylalanine, in decreasing order, were the most effective inhibitors. Results from kinetic studies with amino acids added to gel-filtered urine are consistent with the presence of two AAP isoenzymes with different inhibition characteristics. The ten amino acids which were tested show the same inhibition kinetics. Differences between amino acids are quantitative.

Increased levels of urinary adenosine deaminase binding protein in children treated with cisplatin or methotrexate.

Levels of adenosine deaminase binding protein (ABP), a renal tubular cell antigen, were determined by enzyme immunoassay in urine specimens from seven children with solid tumors who were receiving the recognized nephrotoxins cisplatin or methotrexate. ABP excretion was uniformly increased within the first 3 days after administration of either drug. Elevated ABP levels were usually accompanied by increased excretion of the urinary enzymes N-acetyl-beta-D-glucosaminidase and alanine aminopeptidase. In alkaline urine specimens associated with methotrexate therapy, ABP levels were increased whereas enzyme activities appeared to be unstable. Hence, immunochemical measurement of urinary ABP levels may be adjunctively useful for clinical studies of renal tubular damage.