Organic anion transporter 5 renal expression and urinary excretion in rats with vascular calcification

It has been described renal damage in rats with vascular calcification. The organic anion transporter 5 (Oat5) is only expressed in kidney, and its urinary excretion was proposed as potential early biomarker of renal injury. The aim of this study was to evaluate the Oat5 renal expression and its urinary excretion in an experimental model of vascular calcification in comparison with traditional markers of renal injury. Vascular calcification was obtained by the administration of an overdose of vitamin D3 (300,000 IU/kg, b.w., i.m.) to male Wistar rats. Oat5 urinary abundance was evaluated by Western blotting. Traditional markers of renal injury, such as creatinine and urea plasma levels, urinary protein levels, and urinary alkaline phosphatase (AP) activity, were determined using commercial kits. Histology was assessed by hematoxylin/eosin staining. Oat5 renal expression was evaluated by Western blotting and by immunohistochemistry. An increased expression of Oat5 in renal homogenates, in apical membranes, and in its urinary excretion was observed in rats with vascular calcification. The traditional parameters used to evaluate renal function were not modified, with the exception of histology. It is possible to postulate the urinary excretion of Oat5 as a potential noninvasive biomarker of renal injury associated with vascular calcification.Fil: Hazelhoff, Maria Herminia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bulacio, Romina Paula. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Torres, Adriana Monica. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Organic Anion Transporter 5 (Oat5) Urinary Excretion Is a Specific Biomarker of Kidney Injury: Evaluation of Urinary Excretion of Exosomal Oat5 after N-Acetylcysteine Prevention of Cisplatin Induced Nephrotoxicity

Cisplatin is a commonly used chemotherapeutic agent. Its main side-effect is nephrotoxicity. It was reported that the organic anion transporter 5 (Oat5) urinary excretion is elevated, implying renal perturbation, when no modifications of traditional markers of renal damage are still observed in cisplatin-induced acute kidney injury (AKI). It was also demonstrated that Oat5 is excreted in urine by the exosomal pathway. This study was designated to demonstrate the specific response of the urinary excretion of exosomal Oat5 to kidney injury independently of other cisplatin toxic effects, in order to strengthen Oat5 urinary levels as a specific biomarker of AKI. To accomplish that aim, we evaluated if urinary excretion of exosomal Oat5 returns to its basal levels when cisplatin renal damage is prevented by the coadministration of the renoprotective compound N-acetylcysteine. Four days after cisplatin administration, AKI was induced in cisplatin-treated male Wistar rats (Cis group), as it was corroborated by increased urea and creatinine plasma levels. Tubular damage was also observed. In cotreated animals (Cis + NAC group), plasma urea and creatinine concentrations tended to return to their basal values, and tubular damage was improved. Urinary excretion of exosomal Oat5 was notably increased in the Cis group, but when renal injury was ameliorated by N-acetylcysteine coadministration, that increase was undetected. So, in this work we observed that urinary excretion of exosomal Oat5 was only increased if renal insult is produced, demonstrating its specificity as a renal injury biomarker.Fil: Bulacio, Romina Paula. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Anzai, Naohiko. Dokkyo Medical University; JapónFil: Ouchi, Motoshi. Dokkyo Medical University; JapónFil: Torres, Adriana Monica. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Excreción urinaria del transportador de aniones orgánicos 5 (Oat5) en nefrotoxicidad inducida por cisplatino : potencial biomarcador temprano de esta patología

La insuficiencia renal aguda (IRA) es un síndrome clínico complejo que se caracteriza por una rápida e importante disminución de la función excretora del riñón. A pesar de los avances en el campo de la nefrología en los últimos años, su incidencia sigue siendo preocupantemente alta. Los marcadores que tradicionalmente se emplean para diagnosticar a la IRA, tales como los niveles plasmáticos de urea y creatinina, resultan poco sensibles y específicos, y sus valores se encuentran alterados recién cuando ya existe un significativo daño renal. A consecuencia de esto surge la necesidad de hallar nuevos biomarcadores de daño que permitan realizar un diagnóstico más temprano de la IRA, para mejorar así el pronóstico del paciente. El transportador de aniones orgánicos 5, Oat5 (Slc22a19), se expresa exclusivamente en riñón en la membrana apical de las células de los túbulos proximales, principalmente en la región S3. Nuestro grupo ha sido pionero en la detección de Oat5 en orina. A raíz de ello, posteriores estudios demostraron que la excreción urinaria de Oat5 podría ser considerado como un novedoso biomarcador temprano de daño renal en modelos de IRA isquémica e inducida por cloruro mercúrico. El cisplatino (cis-diaminodicloroplatino II) es un fármaco antineoplásico ampliamente utilizado en la actualidad para el tratamiento de una gran variedad de tumores sólidos. A pesar de su gran efectividad, su uso muchas veces se ve limitado por la nefrotoxicidad que este fármaco produce, principalmente en forma de IRA. En base a todo lo expuesto entonces, el objetivo de este trabajo de Tesis consistió en evaluar si los niveles urinarios de Oat5 también podrían ser utilizados como un biomarcador temprano de daño renal de IRA inducida por cisplatino. En una primera instancia, se analizó el comportamiento de la excreción urinaria de Oat5 en ratas Wistar macho adultas tratadas con una dosis nefrotóxica de cisplatino de 10 mg/kg p.c., i.p., con dos días de tratamiento. Con ello, se pudo corroborar el desarrollo de un cuadro de IRA, ya que parámetros tradicionales utilizados para analizar la función renal se hallaron significativamente modificados en los animales tratados. Por otra parte, los niveles de Oat5 en orina se encontraron notablemente aumentados en el grupo tratado con respecto a los animales controles. En base a estos últimos resultados, la excreción urinaria de Oat5 podría ser propuesta como un marcador de daño renal en IRA inducida por cisplatino. En estudios dosis-respuesta, se pudo observar que la excreción urinaria de Oat5 aumentó de manera dosis-dependiente de cisplatino, y que además se halló incrementada a una dosis tan baja como ser de 1 mg/kg p.c., i.p. del antineoplásico, dos días luego de su administración, cuando aún no se evidenciaban otro tipo de alteraciones renales que se verían manifestadas a mayores tiempos de tratamiento. El comportamiento de la excreción urinaria de Oat5 también fue analizada en el tiempo luego de la administración del cisplatino, para intentar establecer una relación con la progresión del daño renal en un modelo de IRA inducida por dicho antineoplásico. Al respecto, pudo evidenciarse que los niveles de Oat5 en orina aumentaron a los dos días de tratamiento, antes de que se observaran alteraciones en parámetros tradicionales indicadores de daño renal. Sin embargo, los niveles urinarios de dicho transportador no acompañaron a los cambios observados en el tiempo en la función renal. Estos resultados entonces estarían indicando que la excreción urinaria de Oat5 resultaría un biomarcador altamente sensible y temprano de disfunción renal ocasionado por la administración de cisplatino, pero no resultaría útil para realizar un seguimiento de las modificaciones de la función renal en el tiempo en la IRA inducida por cisplatino. Luego, se evaluó si los niveles de Oat5 en orina son sensibles a las modificaciones del estrés oxidativo y/o restauración de la función renal en animales con nefrotoxicidad inducida por cisplatino. Para ello, se analizó el efecto de la N-acetilcisteína (NAC), un compuesto con propiedades antioxidantes, sobre la excreción urinaria de Oat5 en ratas tratadas con cisplatino. En dichos experimentos, pudo observarse que en los animales cotratados con el antineoplásico y la NAC no se evidenció el importante incremento en la excreción urinaria de Oat5 hallado en los animales tratados sólo con cisplatino. Por otra parte, en posteriores experimentos se detectó por primera vez a Oat5 en exosomas urinarios, lo que postula a la vía de excreción exosomal como una de las principales vía involucradas en la eliminación de Oat5 por orina. Con los resultados obtenidos en el presente trabajo de Tesis entonces, se logró caracterizar y aportar mayores conocimientos al comportamiento de la excreción urinaria de Oat5 ante el desarrollo de daño renal causado por un agente nefrotóxico, y permitiría así postular a los niveles urinarios de Oat5 como un novedoso biomarcador temprano y no invasivo de IRA inducida por cisplatino.Fil: Bulacio, Romina Paula. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Ciencias Fisiológicas. Área Farmacología; Argentina

Organic anion transporter 5 (Oat5) renal expression and urinary excretion in rats treated with cisplatin: a potential biomarker of cisplatin-induced nephrotoxicity

Cisplatin is one of the most potent chemotherapeutic antitumor drugs used in the treatment of a wide range of solid tumors. Its primary dose-limiting side effect is nephrotoxicity. The organic anion transporter 5 (Oat5) is exclusively localized in the kidney. Oat5 urinary excretion was recently proposed as a potential early biomarker of acute kidney injury (AKI). The aim of this study was to evaluate Oat5 renal expression and its urinary excretion in rats exposed to different doses of cisplatin, in comparison with traditional markers of renal injury, like renal histology, creatinine and urea plasma levels, creatinine clearance, protein and glucose urinary levels and urinary alkaline phosphatase (AP) activity. Male Wistar rats were treated with a single injection of cisplatin at different doses of 1, 2, 5 and 10 mg/kg b.w., i.p. (Cis1, Cis2, Cis5 and Cis10, n = 4, respectively) and experiments were carried out 48 h after cisplatin administration. The renal expression of Oat5 was evaluated by immunohistochemistry and Western blotting. Oat5 abundance, AP activity, creatinine, glucose and proteins were assayed in urine. Creatinine clearance and creatinine and urea plasma levels were also evaluated. In this experimental model, plasma urea and creatinine levels, creatinine clearance, AP urinary activity and protein and glucose urinary levels were significantly modified only at the highest cisplatin dose of 10 mg/kg b.w., i.p., as compared to control rats. In contrast, Oat5 urinary abundance was increased in a dose-related manner after the administration of cisplatin. Oat5 urinary abundance was elevated at a dose as low as 1 mg/kg b.w., i.p., implying renal perturbation, when no modifications of traditional markers of renal injury are yet observed. Oat5 renal expression was decreased in a dose-related manner, both in homogenates and apical membranes from cisplatin-treated kidneys. The increase in urinary Oat5 excretion might explain the decrease in the amount of Oat5 molecules in the renal tubule cells. Hence, the preclinical animal results showed in this work propose that Oat5 urinary excretion might potentially serve as a non-invasive early biomarker of cisplatin-induced AKI.Fil: Bulacio, Romina Paula. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Torres, Adriana Monica. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Caveolin-2 in urine as a novel biomarker of renal recovery after cisplatin induced nephrotoxicity in rats

Acute kidney injury (AKI) is a heterogeneous clinical syndrome with diverse outcomes. The recovery from AKI has prognostic importance. Little research has been done in order to find biomarkers that can predict recovery from AKI. Cav-2 is one of the main constituents of caveolae and is expressed in kidney. This study analyzed the time course of Cav-2 urinary excretion and renal expression in rats treated with cisplatin. Male Wistar rats were injected with cisplatin (5 mg/kg b.w., i.p.), and the studies were performed after 2, 4 and 14 days. Cav-2 abundance was evaluated in urine, in renal homogenates and in apical membranes by Western blotting. Cav-2 in urine was increased only 14 days after treatment, in the recovery phase of cisplatin-induced AKI. These results show that Cav-2 in urine could be useful as a biomarker of renal recovery, but not as an early biomarker of cisplatin-induced AKI. Cav-2 expression in total renal homogenates was not modified with treatment, but a down-regulation of Cav-2 in apical membranes was observed in treated animals. We hypothesize that Cav-2 internalizes into renal cells from their apical membrane in response to cisplatin, and regulates in this manner different signaling proteins involved in the physiopathology of renal damage.Fil: Bulacio, Romina Paula. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Ciencias Fisiológicas. Area Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Torres, Adriana Monica. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Ciencias Fisiológicas. Area Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentin

Novel Finding of Urinary Erythropoietin as an Early Biomarker of Cisplatin-Induced Nephrotoxicity

Cisplatin is a chemotherapeutic drug used to treat a great variety of solid tumors. Its dose is commonly limited by its nephrotoxicity, manifested as acute kidney injury (AKI). Erythropoietin (Epo) is a glycoprotein hormone that regulates the production of red blood cells. This study was performed to evaluate the presence of endogenous Epo in male Wistar rat urine and to analyse changes in urinary Epo levels in response to cisplatin- induced AKI. Dose-dependent studies and time-dependent experiments were performed to evaluate changes in urea nitrogen and creatinine in plasma as well as Epo, neutrophil gelatinase-associated lipocalin (NGAL), alkaline phosphatase (AP) activity, creatinine and total proteins in urine at 2 days post-dosing. Rats received 2, 5 or 10 mg/kg b.w., i.p. of cisplatin. At 5 mg/kg b.w., i.p. cisplatin, significant increases in urinary Epo were detected. Significant increases in urea nitrogen and creatinine in plasma, NGAL, AP, proteins, and Epo were observed in urine from rats that received 10 mg/kg b.w., i.p. of cisplatin. In the time-dependent experiments, rats were injected with a dose of 5 mg/kg b.w., i.p. of cisplatin, and sampling occurred 2, 4, and 14 days post-dosing. In these animals, there were significant increases in urea nitrogen and creatinine in plasma and total proteins, AP activity, Epo, and NGAL in urine on day 4. Urinary Epo was also detected on day 2. Taken together, these findings provide weight of evidence for urinary Epo as a promising early biomarker of cisplatin-induced AKI in male rats.Fil: Bulacio, Romina Paula. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Ciencias Fisiológicas. Area Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Torres, Adriana Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Ciencias Fisiológicas. Area Farmacología; Argentin

Time course of organic anion transporter 5 (Oat5) urinary excretion in rats treated with cisplatin: a novel urinary biomarker for early detection of drug-induced nephrotoxicity

Cisplatin is a widely used citostatic drug employed in the treatment of many solid tumors. Its principal side-effect is nephrotoxicity. The organic anion transporter 5 (Oat5) is exclusively expressed in the kidneys. The aim of this study was to evaluate the time course of Oat5 urinary excretion and changes in conventional biomarkers, such as creatinine and urea plasma levels (Urp and Crp), and protein and glucose urinary levels (Pu and Gluu), between others, and compared them to the onset and progression of histological changes after cisplatin treatment. Male Wistar rats were treated with cisplatin with 5 mg/kg b.w., i.p., and experiments were carried out after 2, 4, 7 and 14 days of treatment. Two days after cisplatin administration, only Oat5 urinary excretion was found markedly modified. On day 4, Urp, Crp, PU and GluU were increased. By the seventh day, a severe impairment in tubular architecture was observed, and from this point and thereon, Oat5 urinary excretion and PU showed a tendency to return to their basal values. Meanwhile, Urp, Crp and GluU tended to return to their basal values by the day 14 of treatment, when kidney morphology showed an important recovery. So Oat5 urinary abundance was elevated 2 days after cisplatin treatment, when no modifications of traditional markers of renal injury were still observed. Therefore, the results showed in this work, in addition to previous data obtained by our group, propose that Oat5 urinary excretion might potentially serve as a noninvasive early biomarker of cisplatin-induced nephrotoxicity.Fil: Bulacio, Romina Paula. Universidad Nacional de Rosario. Facultad de Cs.bioquimicas y Farmaceuticas. Departamento de Cs.fisiologicas. Area Farmacologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnológico Rosario; ArgentinaFil: Torres, Adriana Monica. Universidad Nacional de Rosario. Facultad de Cs.bioquimicas y Farmaceuticas. Departamento de Cs.fisiologicas. Area Farmacologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnológico Rosario; Argentin

Renal tubular response to titanium dioxide nanoparticles exposure

Titatinum dioxide nanoparticles (TiO2-NPs) are frequently used in several areas. Titanium alloys are employed in orthopedic and odontological surgery (such as hip, knee, and teeth implants). To evaluate the potential acute toxic effects of titanium pieces implantations and in other sources that allow the systemic delivery of titanium, parenteral routes of TiO2-NPs administration should be taken into account. The present study evaluated the impact of subcutaneous administration of TiO2-NPs on renal function and structure in rats. Animals were exposed to a dose of 50 mg/kg b.w., s.c. and sacrificed after 48 h. Titanium levels were detected in urine (135 ± 6 ηg/mL) and in renal tissue (502 ± 40 ηg/g) employing inductively coupled plasma mass spectrometry. An increase in alkaline phosphatase activity, total protein levels, and glucose concentrations was observed in urine from treated rats suggesting injury in proximal tubule cells. In parallel, histopathological studies showed tubular dilatation and cellular desquamation in these nephron segments. In summary, this study demonstrates that subcutaneous administration of TiO2-NPs causes acute nephrotoxicity evidenced by functional and histological alterations in proximal tubule cells. This fact deserves to be mainly considered when humans are exposed directly or indirectly to TiO2-NPs sources that cause the systemic delivery of titanium.Fil: Hazelhoff, Maria Herminia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Ciencias Fisiológicas. Area Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Bulacio, Romina Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Ciencias Fisiológicas. Area Farmacología; ArgentinaFil: Torres, Adriana Monica. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Ciencias Fisiológicas. Area Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentin

Trimetazidine Protects from Mercury-Induced Kidney Injury

Introduction: The presence of mercury in the environment is a worldwide concern. Inorganic mercury is present in industrial materials, is employed in medical devices, is widely used in batteries, is a component of fluorescent light bulbs, and it has been associated with human poisoning in gold mining areas. The nephrotoxicity induced by inorganic mercury is a relevant health problem mainly in developing countries. The primary mechanism of mercury toxicity is oxidative stress. Trimetazidine (TMZ) is an anti-ischemic drug, which inhibits cellular oxidative stress, eliminates oxygen-free radicals, and improves lipid metabolism. The aim of this study was to evaluate whether the administration of TMZ protects against mercuric chloride (HgCl2) kidney damage. Methods: Adult male Wistar rats received only HgCl2 (4 mg/kg bw, sc) (Hg group, n = 5) or TMZ (3 mg/kg bw, ip) 30 min before HgCl2 administration (4 mg/kg bw, sc) (TMZHg group, n = 7). Simultaneously, a control group of rats (n = 4) was studied. After 4 days of HgCl2 injection, urinary flow, urea and creatinine (Cr) plasma levels, Cr clearance, urinary glucose, and sodium-dicarboxylate cotransporter 1 (NaDC1) in urine were determined. Lipid peroxidation (MDA) and glutathione (GSH) levels were measured in kidney homogenates. Results: Rats only treated with HgCl2 showed an increase in urea and Cr plasma levels, urinary flow, fractional excretion of water, glucosuria, and NaDC1 urinary excretion as compared with the control group and a decrease in Cr clearance. TMZHg group showed a decrease in urea and Cr plasma levels, urinary flow, fractional excretion of water, glucosuria, NaDC1 urinary excretion, and an increase in Cr clearance when compared to the Hg group. Moreover, MDA and GSH levels observed in Hg groups were decreased and increased, respectively, by TMZ pretreatment. Conclusion: TMZ exerted a renoprotective action against HgCl2-induced renal injury, which might be mediated by the reduction of oxidative stress. Considering the absence of toxicity of TMZ, its clinical application against oxidative damage due to HgCl2-induced renal injury should be considered. The fact that TMZ is commercially available should simplify and accelerate the translation of the present data “from bench to bedside.” In this context, TMZ become an interesting new example of drug repurposing.Fil: Hazelhoff, Maria Herminia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Ciencias Fisiológicas. Área Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Bulacio, Romina Paula. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Ciencias Fisiológicas. Área Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Torres, Adriana Mónica. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Ciencias Fisiológicas. Área Farmacología; Argentin