Glutamil aminopeptidasa en orina, en microvesículas y en exosomas como marcador de la nefrotoxicidad inducida por el cisplatino en ratas

El objetivo principal de esta Tesis Doctoral es investigar si la excreción de glutamil aminopeptidasa (GluAp) es un marcador de la nefrotoxicidad inducida por el cisplatino en ratas. En un primer experimento se comprobó que la excreción temprana de GluAp, determinada por fluorimetría cinética, ELISA e inmunoblotting se encontraba aumentada en las ratas tratadas con cisplatino, y tenía un valor predictivo sobre la disfunción renal. En otro trabajo se demostró que la GluAp presente en las fracciones microvesicular y exosómica urinarias también correlacionaba con la disfunción renal, tanto si los datos se normalizaban por excreción total diaria, por concentración de creatinina urinaria o por concentración de proteína total en cada fracción. Se concluye que la excreción de GluAp en orina es un marcador del daño renal producido por el cisplatino en ratas que puede ser útil para evaluar el daño tubular renal de manera independiente a la excreción de creatinina.The main objective of this Doctoral Thesis is to investigate whether the excretion of glutamyl aminopeptidase (GluAp), is a marker of cisplatin-induced nephrotoxicity in rats. In a first experiment, the early excretion of GluAp as determined by kinetic fluorimetry, ELISA and immunoblotting was found to be increased in cisplatin-treated rats and it showed a predictive value over renal dysfunction. In other work, it was demonstrated that GluAp in microvesicular and exosomal urinary fractions was also correlated with renal dysfunction if data were normalized by daily total excretion, by concentration of urinary creatinine or by total protein in each fraction. It is concluded that excretion of GluAp in urine is a marker of renal damage induced by cisplatin in rats that can be useful to evaluate renal tubular damage independently of creatinine excretion.Tesis Univ. Jaén. Departamento de Ciencias de la Salud. Leída el 7 de julio de 2017

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-β-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 (p0.259) 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.This study was supported by a grant (R1/12/2010/66) from the University of Jaén with the participation of Caja Rural of Jaén, and from the Carlos III Health Institute of the Spanish Ministry of Health and Consumer Affairs (Red de Investigación Renal, REDinREN RD06/0016/0017 and RD07/0016/2008), “FEDER una manera de hacer Europa.

Thyroid hormones stimulate L-arginine transport in human endothelial cells.

Thyroid hormone activity is associated with L-arginine metabolism and nitric oxide (NO) production, which participate in the cardiovascular manifestations of thyroid disorders. L-arginine transporters play an important role in activating L-arginine uptake and NO production. However, the effects of thyroid hormones on L-arginine transporters in endothelial cells have not yet been evaluated. The following methods were used. We measured L-arginine uptake, mRNA expression of L-arginine transporters, endothelial nitric oxide synthase (eNOS) mRNA and NO generation after the administration of T3, T4 and the T3 analog, 3,3′,5-triiodothyroacetic acid TRIAC in human umbilical vein endothelial cells (HUVECs). We also analyzed the role of αvβ3 integrin and of phosphatidyl-inositol-3 kinase (PI3K), mitogen-activated protein kinases (MAPKs: ERK1/2, p38 and SAPK-JNK) and intracellular calcium signaling pathways as underlying mechanisms. To this end, αvβ3 integrin was pharmacologically inhibited by tetraiodothyroacetic acid (TETRAC) or genetically blocked by silencing αv mRNA and PI3K, MAPKs and intracellular calcium by selective inhibitors. The following results were obtained. Thyroid hormones and the T3 analog TRIAC increased L-arginine uptake in HUVECs, the sodium-independent y+/CAT isoforms, except CAT2b, sodium-dependent y+L system and sodium-independent system b0,+L-arginine transporters, eNOS mRNA and NO production. These effects were suppressed by αvβ3 integrin inhibition with TETRAC or αv integrin downregulation or by PI3K, MAPK or intracellular Ca2+ signaling inhibitors. In conclusion, we report for the first time that activation of L-arginine uptake by thyroid hormones is related to an upregulation of L-arginine transporters. This effect seems to be mediated by activation of αvβ3 integrin receptor and subsequent PI3K, MAPK and intracellular Ca2+ signaling pathways

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

This study was supported by the grants PI13/02743, PI13/02384, and PI18/01715 from the Carlos III Health Institute of Spain, and the Red de Investigacion Renal REDinREN RD16/0009/0033. "FEDER una manera de hacer Europa."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.Instituto de Salud Carlos III PI13/02743 PI13/02384 PI18/01715Red de Investigacion Renal REDinREN "FEDER una manera de hacer Europa" RD16/0009/003

5-aminoisoquinoline improves renal function and fibrosis during recovery phase of cisplatin-induced acute kidney injury in rats.

The aim of the present study is to analyze the effects of 5-aminoisoquinoline (5-AIQ), a poly(ADP-ribose) polymerase-1 (PARP1) inhibitor, over renal dysfunction and fibrosis during recovery phase of cisplatin (CisPt)-induced acute kidney injury (AKI) in rats. Male Wistar rats were distributed in three groups (n=8 each group): control, CisPt, and CisPt + 5-AIQ. Control and CisPt groups received a subcutaneous injection of either saline or 7 mg/kg CisPt, respectively. CisPt + 5-AIQ group received two intraperitoneal injections of 10 mg/kg 5-AIQ 2 h before and 24 h after CisPt treatment. Thirteen days after the treatment, rats were housed in metabolic cages and 24-h urine collection was made. At day 14, CisPt-treated rats showed increased diuresis, N-acetyl-β-d-glucosaminidase (NAG) excretion, glucosuria and sodium fractional excretion (NaFE), and decreased creatinine clearance (CrCl). 5-AIQ significantly increased CrCl and decreased NAG excretion, glucosuria, and NaFE. In plasma, CisPt increased sodium, urea, and creatinine concentrations, while 5-AIQ treatment decreased these variables to the levels of control group. 5-AIQ completely prevented the body weight loss evoked by CisPt treatment. CisPt also induced an increased renal expression of PAR polymer, α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), and collagen-IV. These variables were decreased in CisPt + 5-AIQ group. Tubular lesions and renal fibrosis were also decreased by 5-AIQ treatment. We conclude that inhibition of PARP1 with 5-AIQ can attenuate long-term nephrotoxic effects associated with the CisPt treatment, preventing renal dysfunction and body weight decrease and ameliorating tubular lesions and collagen deposition

Effect of 5-AIQ in cisplatin-treated rats

The aim of this study is to analyze the effects of 5-aminoisoquinoline (5-AIQ), a poly(ADP-ribose) polymerase-1 (PARP1) inhibitor, over renal dysfunction and fibrosis during recovery phase of cisplatin-induced acute kidney injury (AKI) in rats. Male Wistar rats were distributed in three groups (n=8 each group): control, cisplatin (CisPt) and CisPt+5-AIQ. Control and CisPt groups received a subcutaneous injection of either saline or 7 mg/kg cisplatin, respectively. CisPt+5-AIQ group received two intraperitoneal injections of 10 mg/kg 5-AIQ 2 hours before and 24 hours after cisplatin treatment. 13 days after treatment, rats were housed in metabolic cages and 24-h urine collection was made. At day 14, cisplatin-treated rats showed increased diuresis, Nacetyl- β-D-glucosaminidase (NAG) excretion, glucosuria and sodium fractional excretion, and decreased creatinine clearance (CrCl). 5-AIQ significantly increased CrCl and decreased NAG excretion, glucosuria and sodium fractional excretion. In plasma, cisplatin increased sodium, urea and creatinine concentration, while 5-AIQ treatment decreased these variables to the levels of control group. 5-AIQ completely prevented the body weight loss evoked by cisplatin treatment. Cisplatin also induced an increased renal expression of PAR polymer, α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1) and collagen-IV. These variables were decreased in CisPt+5-AIQ group. Tubular lesions and renal fibrosis were also decreased by 5-AIQ treatment. We conclude that inhibition of PARP1 with 5-AIQ can attenuate long-term nephrotoxic effects associated with cisplatin treatment, preventing renal dysfunction and body weight decrease, and ameliorating tubular lesions and collagen deposition.This work was supported by the Carlos III Health Institute of Spain [grant numbers PI13/02743, PI13/02384]; the Red de Investigación Renal REDinREN [grant number RD16/0009/0033]; ‘FEDER una manera de hacer Europa’

Effect of 5-AIQ in cisplatin-treated rats

The aim of this study is to analyze the effects of 5-aminoisoquinoline (5-AIQ), a poly(ADP-ribose) polymerase-1 (PARP1) inhibitor, over renal dysfunction and fibrosis during recovery phase of cisplatin-induced acute kidney injury (AKI) in rats. Male Wistar rats were distributed in three groups (n=8 each group): control, cisplatin (CisPt) and CisPt+5-AIQ. Control and CisPt groups received a subcutaneous injection of either saline or 7 mg/kg cisplatin, respectively. CisPt+5-AIQ group received two intraperitoneal injections of 10 mg/kg 5-AIQ 2 hours before and 24 hours after cisplatin treatment. 13 days after treatment, rats were housed in metabolic cages and 24-h urine collection was made. At day 14, cisplatin-treated rats showed increased diuresis, Nacetyl- β-D-glucosaminidase (NAG) excretion, glucosuria and sodium fractional excretion, and decreased creatinine clearance (CrCl). 5-AIQ significantly increased CrCl and decreased NAG excretion, glucosuria and sodium fractional excretion. In plasma, cisplatin increased sodium, urea and creatinine concentration, while 5-AIQ treatment decreased these variables to the levels of control group. 5-AIQ completely prevented the body weight loss evoked by cisplatin treatment. Cisplatin also induced an increased renal expression of PAR polymer, α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1) and collagen-IV. These variables were decreased in CisPt+5-AIQ group. Tubular lesions and renal fibrosis were also decreased by 5-AIQ treatment. We conclude that inhibition of PARP1 with 5-AIQ can attenuate long-term nephrotoxic effects associated with cisplatin treatment, preventing renal dysfunction and body weight decrease, and ameliorating tubular lesions and collagen deposition.This work was supported by the Carlos III Health Institute of Spain [grant numbers PI13/02743, PI13/02384]; the Red de Investigación Renal REDinREN [grant number RD16/0009/0033]; ‘FEDER una manera de hacer Europa’

ROC curves for urinary aminopeptidase activities at day 1.

<p>ROC curves showing specificity and sensitivity for AlaAp (A), GluAp (B), CysAp (C) and AspAp (D) urinary activities at the first day of treatment.</p

SCr and body weight.

<p>SCr concentration (mg/dl) (A) and body weight increase (%) in Control and Cisplatin groups at the end of the experiment. Individual samples and means ± SEM are displayed; * p<0.05, **p<0.001 Cisplatin <i>vs</i>. Control (n = 10 each group).</p