Urine Angiotensin II Signature Proteins as Markers of Fibrosis in Kidney Transplant Recipients.

BACKGROUND: Interstitial fibrosis/tubular atrophy (IFTA) is an important cause of kidney allograft loss; however, noninvasive markers to identify IFTA or guide antifibrotic therapy are lacking. Using angiotensin II (AngII) as the prototypical inducer of IFTA, we previously identified 83 AngII-regulated proteins in vitro. We developed mass spectrometry-based assays for quantification of 6 AngII signature proteins (bone marrow stromal cell antigen 1, glutamine synthetase [GLNA], laminin subunit beta-2, lysophospholipase I, ras homolog family member B, and thrombospondin-I [TSP1]) and hypothesized that their urine excretion will correlate with IFTA in kidney transplant patients. METHODS: Urine excretion of 6 AngII-regulated proteins was quantified using selected reaction monitoring and normalized by urine creatinine. Immunohistochemistry was used to assess protein expression of TSP1 and GLNA in kidney biopsies. RESULTS: The urine excretion rates of AngII-regulated proteins were found to be increased in 15 kidney transplant recipients with IFTA compared with 20 matched controls with no IFTA (mean log2[fmol/µmol of creatinine], bone marrow stromal cell antigen 1: 3.8 versus 3.0, P = 0.03; GLNA: 1.2 versus -0.4, P = 0.03; laminin subunit beta-2: 6.1 versus 5.4, P = 0.06; lysophospholipase I: 2.1 versus 0.6, P = 0.002; ras homolog family member B: 1.2 versus -0.1, P = 0.006; TSP1_GGV: 2.5 versus 1.9; P = 0.15; and TSP1_TIV: 2.0 versus 0.6, P = 0.0006). Receiver operating characteristic curve analysis demonstrated an area under the curve = 0.86 for the ability of urine AngII signature proteins to discriminate IFTA from controls. Urine excretion of AngII signature proteins correlated strongly with chronic IFTA and total inflammation. In a separate cohort of 19 kidney transplant recipients, the urine excretion of these 6 proteins was significantly lower following therapy with AngII inhibitors (P < 0.05). CONCLUSIONS: AngII-regulated proteins may represent markers of IFTA and guide antifibrotic therapies.

Acute, nonfatal intoxication with trichloroethylene.

Nonfatal acute inhalation of trichloroethylene (TRI) at work was described. The subject, male, 54 years old, was drawn unconscious by a metal-degreasing machine and immediately sheltered in intensive care unit. Other than basic life support and common laboratory indices, blood and urine were collected to measure dose and kidney effect parameters such as TRI in blood and urine, trichloroethanol (TCE) and trichloroacetic acid (TCA) in urine, and total urinary proteins (TUP), urinary glutamine synthetase (GS) and urinary N-acetyl-beta-D-glucosaminidase (NAG). Two hours after accident, TRI in blood was 9 mg/l, but after 38 h it was below 1 mg/l. TCE and TCA have a peak 11 and 62 h after poisoning, respectively. Acute renal involvement was revealed by a peak of urinary proteins and enzymes 7 h after exposure with a second peak 74 h after. Seven day after hospitalisation the patient was dismissed with complete recovery. This nonfatal intoxication with TRI shows that the exposure was approximately 150 ppm, three times the ACGIH TLV (50 ppm) and that kidney was the only organ affected. Urinary enzymes, in particular GS, are good indices to monitor transient effects of TRI on the kidney.

Historical control data on urinary and renal tissue biomarkers in naive male Wistar rats.

Total proteins, angiotensin-converting enzyme, N-acetyl-beta-D-glucosaminidase, glutamine transaminase K and glutamine synthetase were determined in urine collected overnight (14 h: 6:00 p.m.-8:00 a.m.) from naive male Wistar rats; glutamine transaminase K and glutamine synthetase in the kidney 10,000 g supernatant and p-aminohippurate uptake in renal cortical slices also were measured. Urinary parameters were related both to urinary creatinine concentration and urinary flow rate; kidney parameters were related to protein concentration (enzymes) or slice/medium (S/M) ratio (p-aminohippurate uptake). The following reference ranges (1.0 and 99.0 percentiles) were obtained: urine: total urinary proteins (195 samples) 0.03-0.29 g mmol(-1) creatinine and 0.13-1.77 mg h(-1); angiotensin-converting enzyme (115 samples) 8.9-63.7 micromol mmol(-1) creatinine and 59.4-282.7 nmol h(-1); glutamine transaminase K (115 samples) 0-1.7 micromol mmol(-1) creatinine and 0-8.5 nmol h(-1); N-acetyl-beta-D-glucosaminidase (72 samples) 0.7-5.0 micromol mmol(-1) creatinine and 4.9-28.4 nmol h(-1) (naive male rats did not excrete glutamine synthetase); kidney: glutamine transaminase K (36 samples) 14.5-32.8 nmol mg(-1) protein; glutamine synthetase (22 samples) 13.9-48.6 nmol mg(-1) protein and p-aminohippurate (54 samples) 4.77-17.89 S/M. Urinary creatinine (r = -0.780), total urinary proteins (r = -0.521), angiotensin-converting enzyme (r = -0.650) and N-acetyl-beta-D-glucosaminidase (r = -0.796) but not glutamine transaminase K were well correlated with diuresis. In addition, the same parameters, but not glutamine transaminase K, were well correlated with creatinine (r = 0.604,0.701 and 0.747, respectively). Significant correlation also was observed between urinary indices adjusted to creatinine or urinary flow rate (total urinary proteins: r = 0.813; angiotensin-converting enzyme: r = 0.677; glutamine transaminase K: r = 0.939; N-acetyl-beta-D-glucosaminidase: r = 0.657). Finally, a low but significant correlation was found between total urinary proteins and angiotensin-converting enzyme (r = 0.293) and N-acetyl-beta-D-glucosaminidase (r = 0.471).

Glutamine synthetase activity in rat urine as sensitive marker to detect S3 segment-specific injury of proximal tubule induced by xenobiotics.

The possibility of detecting segment-specific injury of the proximal tubule by means of urinary enzymes was investigated in rats. Urinary glutamine synthetase, an enzyme exclusively localized in the S3 segment, and N-acetyl-beta-D-glucosaminidase, prevalently a S1-S2, but S3 enzyme also, were determined after single treatment with 100 mg/kg body wt. of hexachloro-1:3-butadiene (HCBD; i.p.), toxic for the S3 segment, or 25 mg/kg body wt. of potassium dichromate (s.c.), toxic for the S1-S2 segments. Excretion of total urinary proteins was also measured. In addition, a dose-response relationship was determined between three doses (50, 100 and 200 mg/kg body wt.) of HCBD and glutamine synthetase activity in urine. Glutamine synthetase activity, measured according to a new assay for urine based on modification of methods developed for organs, increased in the urine only when the S3 segment of the proximal tubule was damaged, as demonstrated by histological findings of the kidneys. HCBD caused early excretion of the enzyme related to the necrosis of the S3 segment, whereas potassium dichromate caused a slight increase only when the resulting lesion to this segment (vacuolization) began to develop. On the contrary, N-acetyl-beta-D-glucosaminidase activity showed the peak of excretion 24 and 34 h after treatment with HCBD or potassium dichromate, respectively, according to the histological findings of necrosis of the S3 segment (the former) and vacuolization of the S1-S2 segments (the latter). Excretion of total urinary proteins reached the peak 24 h (HCBD) and 48 h (potassium dichromate) after treatment. HCBD at 200 mg/kg body wt, caused a peak of glutamine synthetase activity in urine 10 h after injection, whereas the peak caused by doses of 50 and 100 mg/kg body wt. occurred 24 h following treatment. The peak of enzyme activity in urine significantly increased with the dose. The results suggest that the measurement of urinary activity of S3 segment-specific enzyme as glutamine synthetase allows us to detect early S3 segment-specific injury of the proximal tubule. In addition, the method for urinary enzyme activity appears sensitive, simple and fast.