The Long-Term Study of Urinary Biomarkers of Renal Injury in Spontaneously Hypertensive Rats.

BACKGROUND: The age-related increase in blood pressure in spontaneously hypertensive rats (SHRs) is associated to cardiac hypertrophy, heart failure, and renal injury. Here, we investigated for the first time the urinary enzymatic activities of glutamil aminopeptidase (GluAp), alanyl aminopeptidase (AlaAp), dipeptidyl peptidase-4 (DPP4), and Klotho urinary levels, proteins that are strongly expressed in the kidney, as early biomarkers of renal injury in SHRs. METHODS: Male SHR and Wistar Kyoto (WKY) rats were studied from 2 to 8 months old. Systolic blood pressure (SBP), the heart rate (HR), metabolic variables, and urinary markers were measured monthly. At the end of the study, a histopathological evaluation of the kidney was performed. RESULTS: Kidneys of SHR did not develop signs of relevant histopathological changes, but showed increased glomerular area and cellularity. Plasma creatinine was decreased, and creatinine clearance was augmented in SHR at the end of the study. Urinary excretion of Klotho was higher in SHR at 5 and 8 months old, whereas plasma Klotho levels were similar to WKY. GluAp, AlaAp, and DPP4 urinary activities were increased in SHR throughout the time-course study. A positive correlation between glomerular area and cellularity with creatinine clearance was observed. Urinary GluAp, AlaAp, DPP4, and Klotho showed positive correlations with SBP. CONCLUSIONS: GluAp, AlaAp, DPP4, and Klotho in the urine are useful tools for the evaluation of renal damage at early stages, before the whole histopathological and biochemical manifestations of renal disease are established. Moreover, these observations may represent a novel and noninvasive diagnostic approach to assess the evolution of kidney function in hypertension and other chronic diseases.

Glutamyl aminopeptidase in microvesicular and exosomal fractions of urine is related with renal dysfunction in cisplatin-treated rats.

PURPOSE: The aim of this work was to investigate if the content of glutamyl aminopeptidase (GluAp) in microvesicular and exosomal fractions of urine is related with renal dysfunction in cisplatin-treated rats. METHODS: Urine samples were collected 24 hours after injection of cisplatin (7 mg/kg, n = 10) or saline serum (n = 10), and they were subjected to differential centrifugation at 1.000, 17.000 and 200.000 g to obtain microvesicular and exosomal fractions. GluAp was measured with a commercial ELISA kit in both fractions. Serum creatinine (SCr) and body weight were measured 15 days after treatment. We analyzed if early excretion of GluAp in microsomal and exosomal fractions was correlated with final SCr and body weight increase. In a second experiment, enzymatic activities of GluAp and alanyl aminopeptidase (AlaAp) in urine, microvesicular and exosomal fractions were measured three days after injection. We analyzed the correlation of both markers with SCr determined at this point. Finally, we studied the expression of GluAp and extracellular vesicles markers Alix and tumor susceptibility gene (TSG101) in both fractions by immunoblotting. RESULTS: GluAp excretion was increased in all fractions of urine after cisplatin treatment, even if data were normalized per mg of creatinine, per body weight or per total protein content of each fraction. We found significant predictive correlations with SCr concentration, and inverse correlations with body weight increase determined 15 days later. Three days after injection, aminopeptidasic activities were markedly increased in all fractions of urine in cisplatin-treated rats. The highest correlation coefficient with SCr was found for GluAp in microvesicular fraction. Increase of GluAp in microvesicular and exosomal fractions from cisplatin-treated rats was confirmed by immunoblotting. Alix and TSG101 showed different patterns of expression in each fraction. CONCLUSIONS: Determination of GluAp content or its enzymatic activity in microvesicular and exosomal fractions of urine is an early and predictive biomarker of renal dysfunction in cisplatin-induced nephrotoxicity. Measurement of GluAp in these fractions can serve to detect proximal tubular damage independently of glomerular filtration status.

Immunological detection of glutamyl aminopeptidase in urine samples from cisplatin-treated rats.

PURPOSE: The aim of this work is to demonstrate if urinary excretion of glutamyl aminopeptidase (GluAp) can be quantified by immunological methods. EXPERIMENTAL DESIGN: Urine samples from control and cisplatin-treated rats (n = 10 each group) were obtained at 1, 8, and 15 days after cisplatin injection. GluAp was analyzed by kinetic fluorimetry, ELISA, and immunoblotting. Sensitivity and specificity was studied for fluorimetric activity and ELISA 24 h after cisplatin injection. We also analyzed the predictive value over renal dysfunction at the end of the experiment. RESULTS: GluAp was easily detected by immunoblotting and ELISA, and its urinary excretion was increased in cisplatin-treated rats (p < 0.01). Results obtained with ELISA were strongly correlated (r = 0.8186; p < 0.0001) with fluorimetric activity. Kinetic fluorimetry was the method with the highest AUC (AUC = 1) and the highest predictive value over serum creatinine (r = 0.7630; p = 0.0001) and body weight increase (r = -0.8721; p < 0.0001). CONCLUSIONS AND CLINICAL RELEVANCE: GluAp can be detected in urine samples with immunological methods, making possible the development of an antibody-based kit for its determination. Its excretion correlates with the extent of renal dysfunction in cisplatin-treated rats, confirming its value as an early marker of renal damage that can be a diagnostic aid in renal diseases.

Detection of renal and urinary tract proteins before and after spaceflight.

BACKGROUND: The recent evolution of genomics and subsequently proteomics offers a major advance in the ability to understand individual human variation in disease and the molecular level changes induced by certain environmental exposures. This original study examines urinary proteome composition to enable the understanding of molecular homeostatic mechanisms in spaceflight and presents the potential for early detection of subclinical disease, microgravity risk mitigation strategies, and countermeasure development for exploration-class missions. METHODS: The urinary proteome composition of six Russian cosmonauts (men, ages 35-51) who flew long-duration missions of 169-199 d was determined 30 d before flight and compared to repeat studies 1 and 7 d postflight. RESULTS: There were 430 proteins identified. Of those, 15 proteins originated in the renal tissues. Of the 15 urinary proteins, 10 were consistently present in the urine. However, the presence of five of the urinary proteins--neutral endopeptidase (NEP), afamin (AFAM), aquaporin-2 (AQP2), aminopeptidase A (AMPE), and dipeptidyl peptidase 4 (DPP4)--was dependent on spaceflight exposure. DISCUSSION: Proteomic investigation of pre- and postflight urine and bioinformation approaches to proteome analysis provide important data relative the mechanism of kidney function in spaceflight. In this initial study, we determined that the evaluation of urinary proteins may help investigators understand changes that are occurring in microgravity. Once additional ground-based and in-flight data are collected, it is feasible to develop targeted studies for tracking specific spaceflight related changes, determine countermeasure and risk-mitigation effectiveness, and possibly detect subclinical disease in flight crewmembers.