Skip to main content
NCBI home page
Toxicology Research logoLink to Toxicology Research
. 2016 Jan 7;5(2):530–538. doi: 10.1039/c5tx00436e

The urinary excretion of an organic anion transporter as an early biomarker of methotrexate-induced kidney injury

María J Severin a, Mara S Trebucobich a, Patricia Buszniez a, Anabel Brandoni a, Adriana M Torres a,✉
PMCID: PMC6062349  PMID: 30090367

graphic file with name c5tx00436e-ga.jpgOat5 urinary excretion is a novel urinary biomarker for early detection of methotrexate-induced kidney injury.

Abstract

Methotrexate (MTX) belongs to a group of medicines known as antimetabolites. It is commonly used in the treatment of malignant diseases and is prescribed in autoimmune and chronic inflammatory disorders. Along with its effective therapeutic power, MTX has adverse effects on several organs, including the kidney. The organic anion transporter 5 (Oat5) is exclusively localized in the renal apical membrane. Oat5 urinary excretion was proposed as an early biomarker in ischemic and nephrotoxic-induced kidney injury and in renal damage due to vascular calcification in preclinical models. The aim of this study was to evaluate Oat5 renal expression and urinary excretion in rats 48 h after the exposure to different doses of MTX, in comparison with traditional markers of renal injury, such as creatinine and urea plasma levels, protein urinary levels, urinary alkaline phosphatase (AP) activity, fractional excretion of water (FEWater) and renal histology. Male Wistar rats were treated with a single intraperitoneal injection of MTX at different dosages: 40-80-120-180-360 mg per kg b.w. (M40, M80, M120, M180, M360, n = 4, respectively) and experiments were carried out 48 h after MTX administration. Oat5 renal expression was evaluated by western blotting and immunohistochemistry. Traditional parameters were only modified at the higher MTX dose (M360). Conversely, Oat5 urinary excretion was elevated at the middle dose of 80 mg per kg b.w. Oat5 renal expression was modified at the highest dose as well, both in homogenates and in apical membranes. These results suggest that Oat5 urinary excretion might serve as an early biomarker of MTX-induced kidney injury.

1. Introduction

Methotrexate (MTX) belongs to a group of medicines known as antimetabolites. The folic acid antagonist, MTX, is used clinically to inhibit the synthesis of purines and pyrimidines.1,2 MTX is commonly used at doses of 1–33 g m–2 (also termed high-dose MTX, HDMTX) in the treatment of malignant diseases, such as osteosarcoma, primary central nervous system lymphomas, lymphoblastic leukemia as well as adult and pediatric non-Hodgkin lymphomas, breast cancer and head and neck cancer.3–5 At lower doses, MTX is used in the maintenance therapy of acute lymphoblastic leukemia and in the treatment of rheumatoid arthritis, since MTX is the disease-modifying antirheumatic drug of choice worldwide. Likewise, this agent is prescribed in cases of psoriasis and other chronic inflammatory disorders to inhibit inflammatory leukocytes proliferation.6,7 Along with its effective therapeutic power, MTX has adverse effects on several organs, including the kidney. HDMTX therapy may produce acute kidney injury (AKI), thereby impairing its own elimination and increasing its toxicity. AKI is the most common form of renal toxicity due to HDMTX and is seen in 2–10% of treatment cycles. MTX-induced AKI is attributed to tubular obstruction, due to the precipitation of MTX and its metabolites which may be exacerbated by low urinary pH and reduced tubular flow owing to hypovolemia, or via a direct toxic effect in the renal tubules.5,8,9 Enhanced neutrophil infiltration and oxidative stress are likely to contribute to MTX-induced renal damage.10 Up to 90% of MTX is cleared by the kidneys, and renal impairment can result in a delayed drug excretion and subsequently, systemic toxicity. Toxic effects are typically treated through hydration and alkalinization of the patient and through the use of pharmacokinetically guided leucovorin rescue. Despite these preventive measures, MTX-induced nephrotoxicity continues to occur.

The traditional blood (creatinine, blood urea nitrogen) and urinary markers of kidney injury used for diagnosis and prognosis of AKI are insensitive and nonspecific, and do not directly reflect injury to kidney cells but rather delayed functional damage consequences.11,12 Efforts to prevent foreseeable kidney damage, as a product of various pharmacological therapies or disease states, are affected by the delay of the methods currently used to diagnose AKI. Discovery of new biomarkers is required to improve the early detection of AKI and optimize the effectiveness of treatments and prognostic assessment.13 The organic anion transporter 5 (Oat5, Slc22a19) belongs to the organic anion transporter family and has been cloned and characterized.14,15 Its expression remains exclusive to the kidney, more specifically it is located on the apical membrane of the proximal tubule in the straight segment S3, and to a lesser extent in the S2 segment. Oat5 transports ochratoxin A, estrone-3-sulphate, dehydroepiandrosterone sulphate, dicarboxylates (α-ketoglutarate and succinate) and interacts with chemically heterogeneous anionic compounds, such as nonsteroidal anti-inflammatory drugs, diuretics, bromosulfophthalein and penicillin G.15

Our group was pioneering in the detection of Oat5 in urine.16 We have reported an important urinary increase in Oat5 in ischemia, mercury and cisplatin induced renal failure in preclinical murine models, suggesting that urinary Oat5 excretion may serve as a novel potentially valuable biomarker of kidney injury.17–19 In addition, Oat5 abundance in urine has been postulated as a noninvasive biomarker of renal damage associated with vascular calcification, pathology of nonrenal origin but associated with some type of renal nephropathy.20

To nominate a promising compound as a universal biomarker of kidney injury, first of all it might be found altered in several preclinical models of renal pathology. There is a high variability when the same biomarker is used to diagnose even in the same clinical context. In different clinical situations, there are variables, such as sensitivity and specificity that might be modified. Then, it is relevant to study each new biomarker in many diverse preclinical models of renal damage.

Presently, there is no rapid test to recognize MTX-induced renal damage, and when it is detected with traditional markers, renal function and therefore health integrity are highly compromised. This obstacle opens a new gate to study the possible application of Oat5 urinary excretion as an early biomarker of MTX induced nephrotoxicity.

Accordingly, the aim of this study was to evaluate Oat5 renal expression and urinary excretion in rats 48 h after the exposure to different doses of MTX, in comparison with the traditional markers of renal injury, such as creatinine and urea plasma levels, protein urinary levels, urinary alkaline phosphatase activity and renal histology. These studies were performed in order to determine if Oat5 urinary excretion might serve as an early biomarker of MTX-induced kidney injury.

2. Results and discussion

Rats exposed to 40, 80, 120 and 180 mg per kg body weight (b.w.), intraperitoneal (i.p.) of MTX showed no differences in plasma urea and creatinine levels, and in creatinine clearance values as compared to control animals. In contrast, plasma urea and creatinine levels were significantly increased and creatinine clearance was significantly decreased in the M360 group in comparison with control rats as it is shown in Fig. 1. These results reflect renal dysfunction and kidney injury in rats treated with the highest dose of MTX. The treatment with this agent produced a significant decrease in body weight in all the doses evaluated after 48 h of injection (Table 1). However, only the M360 group displayed kidney/body weight ratio levels higher than control animals. Urine output was lower than control rats in the middle doses of 40, 80 and 120 mg per kg of MTX while in the M180 group urine output levels were restored and there was an increase in the M360 group in this parameter.

Fig. 1. Urea (A) and (B) creatinine plasma levels and (C) renal clearance of creatinine in control and treated animals with different doses of MTX, M40 (n = 4), M80 (n = 4), M120 (n = 4), M180 (n = 4) and M360 (n = 4) 48 h before the experiments. Results are expressed as mean values ± SEM. (a) p < 0.05 versus control, (b) p < 0.05 versus M40, (c) p < 0.05 versus M80, (d) p < 0.05 versus M120, (e) p < 0.05 versus M180, (f) p < 0.05 versus M360.

Fig. 1

Open in a new tab

Table 1. Body weight decreased in 48 h of treatment, kidney/body weight ratio and urine volume in control and treated rats with MTX; M40, M80, M120, M180 and M360 after MTX administration.

  Control (n = 4) M40 (n = 4) M80 (n = 4) M120 (n = 4) M180 (n = 4) M360 (n = 4)
Body weight decreased in 48 h (%) 5.04 ± 0.65 7.9 ± 0.58a,d,f 8.65 ± 0.72a,d,f 11.8 ± 1.02a,b,c,f 10.6 ± 0.76a,f 14.21 ± 0.46a,b,c,d,e
Kidney/body weight ratio (×10–3) 7.04 ± 0.08 7.41 ± 0.19c,f 6.56 ± 0.06b,e,f 7.04 ± 0.06f 7.41 ± 0.12c,f 8.10 ± 0.31a,b,c,d,e
Urine volume (mL per min per 100 g) 4.84 ± 0.39 2.23 ± 0.29a,e,f 2.32 ± 0.14a,e,f 2.67 ± 0.12a,e,f 4.77 ± 0.63b,c,d,f 7.13 ± 0.47a,b,c,d,e
Open in a new tab

A histological evaluation of kidneys from rats treated with 40, 80, 120, 180 and 360 mg per kg of MTX was performed. In the M40 and in M80 groups (Fig. 2B) no significant histological damage were observed compared to control kidneys. In contrast the M120 group showed a mild impairment (Fig. 2C) and the M180 and M360 groups showed a growing and more dramatic injury with the increasing MTX administered dose (Fig. 2D–F). As previously described, MTX has a direct toxic effect on renal tubular cells.21,22 Fig. 2 shows tubular desquamation cells, tubular dilatation, necrotic processes, the presence of vacuoles and cell swelling, particularly in the M360 group.

Fig. 2. Optical microscopy photos of kidney histology in control (A), M40 (B), M80 (C), M120 (D), M180 (E) and M360 (F) rats (hematoxylin–eosin staining). In the M40 group no significant histological damage were observed compared to control kidneys. M120, M180 and M360 images show a growing injury with the increasing MTX administered dose: tubular dilatation (arrow) tubular desquamation cells (arrowhead), necrotic processes (cross), vacuoles and cell swelling (star). These pictures are representatives of samples obtained from 4 animals from each experimental group. Bars 40 μm.

Fig. 2

Open in a new tab

Oat5 abundance in urine was related to urinary creatinine concentrations, as previously described17,18 in order to correct for variations in urine production. Potential changes in urinary biomarker concentrations induced by the hydration status and diuretic therapy can occur. A commonly employed correction factor for urinary dilution is to express urinary biomarkers adjusted for urinary creatinine concentration in research studies.23Fig. 3A shows that Oat5 urinary levels were significantly higher in the M120, M180 and M360 groups. However, animals treated with 80 mg per kg of MTX showed a significant augmentation in Oat5 urinary excretion, compared to controls, when tested using a t-test (p < 0.05). As seen in this figure, increase in Oat5 urinary abundance is proportional to the dose of MTX used.

Fig. 3. Oat5 abundance in urine (A), urinary AP activity (B), urine proteins levels (C) and fractional excretion of water (D) in control (n = 4), and treated animals with different doses of MTX, M40 (n = 4), M80 (n = 4), M120 (n = 4), M180 (n = 4) and M360 (n = 4) 48 h before the experiments. For the assay of Oat5 abundance, urine samples were separated by SDS-PAGE and blotted onto nitrocellulose membranes. Densitometric quantification of Oat5 western blotting from urine are expressed as arbitrary units related to urinary creatinine concentration in order to correct for variations in urine production. The mean of the control value was set as 100%. Urinary AP activity and urine proteins levels were determined using commercial kits and were related to urinary creatinine concentration in order to correct for variations in urine production. Results are expressed as mean values ± SEM. (a) p < 0.05 versus control, (b) p < 0.05 versus M40, (c) p < 0.05 versus M80, (d) p < 0.05 versus M120, (e) p < 0.05 versus M180, (f) p < 0.05 versus M360.

Fig. 3

Open in a new tab

Additional parameters to evaluate renal injury and function, such as urinary activity of AP and protein urinary concentration were measured and related to urinary creatinine concentration. In addition, the fractional excretion of water was measured as well. As it is shown in Fig. 3B–D, all these parameters were only significantly increased in urine samples of the M360 group as compared to control animals, indicating that significant renal damage was produced at the higher dose of MTX.

Total renal homogenates and the apical membranes from control and treated animals were subjected to western blotting for Oat5 protein. Oat5 abundance in both total homogenates and apical membranes from the M40, M80, M120, and M180 groups were not different from control ones. On the contrary, Oat5 abundance was significantly lower in the M360 group for both renal homogenates and apical membranes (Fig. 4).

Fig. 4. Western blotting for Oat5 in homogenates (A) and apical membranes (B) (20 μg proteins) from kidneys of control and treated rats with MTX, M40, M80, M120, M180 and M360. Proteins were separated by SDS-PAGE and blotted onto nitrocellulose membranes. The mean of the control value was set as 100%. Results are expressed as mean values ± SEM. (a) p < 0.05 versus control, (b) p < 0.05 versus M40, (c) p < 0.05 versus M80, (d) p < 0.05 versus M120, (e) p < 0.05 versus M180, (f) p < 0.05 versus M360.

Fig. 4

Open in a new tab

Immunohistochemistry studies (Fig. 5) showed a strong Oat5 labeling associated with the apical plasma membrane in proximal tubule cells. Oat5 was consistent with the results observed by western blotting in each experimental group. This study confirmed the reduced expression of Oat5 in kidney from the M360 group. In this experimental group, Oat5 presented an irregular pattern of expression, showing lower labeling in some tubules and similar labeling in others as compared with the staining observed in proximal tubules from control kidneys. A similar pattern of expression was observed for Oat1 and Oat3 (another two members of Oats family) in kidneys from rats with ischemic-AKI.24 Representative Oat5 labeling for control, M80, M180 and M360 groups is shown in Fig. 5.

Fig. 5. Immunohistochemistry for Oat5 in renal tissue from control (A), M80 (B) and M180 (C) and M360 (D) rats. Serial sections from each rat kidney were stained using a non-commercial anti-Oat5 antibody. Oat5 labeling was associated with the apical plasma membranes in proximal tubule cells (arrows). In M360 rats, it can be seen that Oat5 is greatly reduced, with an irregular pattern of expression, showing lower expression in some tubules (arrowheads) and similar expression in others (arrows) as compared with the staining observed in the proximal tubules of control rats. These pictures are representatives of typical samples obtained from 4 animals from each experimental group. Bars 40 μm.

Fig. 5

Open in a new tab

Kidney disease is a global health problem; particularly AKI incidence is increasing.25,26 AKI is the generic term for an abrupt and sustained decrease in renal function resulting in the retention of nitrogenous (urea and creatinine) and non-nitrogenous waste products that occurs within hours to days.27 Common presenting symptoms for patients with AKI include volume depletion (as a result from diarrhea, vomiting or decreased oral intake), shock, anuria, edema, and unilateral flank pain. However, patients can also present renal injury without any clear signs of renal insufficiency, and laboratory evaluation is needed to reveal AKI.28 MTX is an antimetabolite used in the management of certain types of cancer and several auto-immune diseases as well. HDMTX, generally considered ≥500 mg m–2, is commonly employed in regimens for leukemia, lymphoma and osteosarcoma. Unfortunately, HDMTX therapy may produce AKI, thereby impairing its own elimination and increasing its toxicity. The systemic toxicity of HDMTX regimens requires pretreatment urinary alkalinization, intravenous hydration and planned leucovorin rescue, however successful rescue by leucovorin depends on the rapid elimination of MTX by the kidneys.5,29 MTX enters the cell via the reduced folate carrier and the organic anion transport system, primarily Oat3 and Oat1, the last one playing a minor role.30 Once in the cell, this agent undergoes polyglutamylation catalyzed by folylpolyglutamate synthetase. MTX-induced renal dysfunction is believed to be mediated by the precipitation of MTX and its metabolites in the renal tubules, thereby causing tubular obstruction and acute tubular necrosis. Other toxicity mechanisms involve renal hypoperfusion and a direct toxic effect of MTX on the renal tubules with reactive oxygen radical production triggering lipoperoxidation and mitochondrial damage.5,21,22 Previous studies in pigs and rodents treated with MTX, showed necrosis and morphological changes in proximal and distal tubules, the presence of vacuoles and high endocytic activity on the area near the apical membrane of the proximal tubule along with an increase in cell size and organelles.1,21 Notwithstanding, little it is known about MTX nephrotoxicity. AKI due to HDMTX is assessed as an abrupt increase in creatinine and plasma urea, with dysuria and hematuria. Nevertheless, a majority of patients are initially asymptomatic, and most of them show nonoliguric renal dysfunction. Plasma creatinine levels usually return to normal values within 2–3 weeks, although some cases develop severe symptoms and sometimes in an irreversible way.5 In the era of optimal care, an appropriate clinical diagnostic method is not available to detect this pathology on time. Although it is performed as standardized care in medical practice, many severe cases of MTX-induced nephrotoxicity continue to occur. The accuracy of using serum creatinine to predict AKI is severely limited since kidney injury occurs before creatinine levels rise. Because of this disadvantage, new biomarkers have been studied to pursue an earlier detection of this pathology.28 Another standard to detect renal injury are histopathology studies. However, it does not identify non-histopathology-associated types of kidney disturbances, such as inhibition of transporters in the proximal tubule. Moreover, use of histopathology as a benchmark for kidney injury in humans is usually impractical, except in relatively rare instances when a kidney biopsy is justified.31 In kidney diseases it is especially true that one biomarker by itself may not satisfy the requirements of an ideal biomarker. The incorporation of several different markers into a biomarker panel may enable simultaneous assessment of site-specific kidney injury with an indication of the damage degree.11 Particularly, urinary biomarkers have many advantages, including the noninvasive nature of sample collection, the reduced number of interfering proteins, and the increased specificity for kidney injury.

Oat5 is located in the apical membranes of the proximal tubule S3 segment where it functions as a dicarboxylate/organic anion transporter. Our group has proposed urinary excretion of Oat5 as an early and novel biomarker of proximal tubule damage in ischemic, mercuric and cisplatin induced kidney injury.16–19 In the present study, our goal was to determine if Oat5 represents a more sensitive and earlier biomarker of MTX-induced AKI compared to traditional, routinely used biomarkers of nephrotoxicity. A MTX dose–response study was performed. Plasma urea and creatinine levels, AP urinary activity, urinary protein levels and the fractional excretion of water were significantly elevated above control values only at the highest MTX dose of 360 mg per kg b.w., i.p. Histological mild injury was detected in the M120 group and more dramatic damage were observed in M180 and M360 rats. In contrast, urinary Oat5 protein levels increased in a dose-related manner after injection of MTX. The mean increases of urinary Oat5 were 77, 99, 133, 667% higher than the control for MTX doses of 80, 120, 180 and 360 mg per kg b.w., i.p., respectively. No changes were found in the M40 group. Oat5 urinary abundance was elevated at a dose of 80 mg per kg b.w., i.p., allowing predicting renal perturbation, when no modifications of traditional markers of renal injury were still observed.

Our work also evaluates Oat5 renal expression. Results showed a significant decrease of Oat5 both in homogenates and apical membranes from MTX-treated kidneys only in the M360 group. This data suggest a decrease in Oat5 synthesis or an increase in its degradation, probably due to the tubular damage caused by MTX at a very high dose. Since the expression of this protein was not modified in homogenates and in apical membranes in the other groups of treated animals, it is possible to suggest that after a lower toxicant insult, Oat5 urinary excretion is dependent on a selective apical pathway.

Drugs with different mechanisms of toxicity frequently affect different parts of the kidney. Most drug-induced renal injuries affect the proximal tubules. However, drug toxicity initially targeted to the glomerulus or more distal parts of the nephron may also cause secondary injury to proximal tubules. The detection of proximal tubule injury might thus provide a sensitive way to monitor most, but not all, toxicities.31 Bibliography proposes that MTX mainly produces its toxic effect on distal tubules by its own precipitation or its metabolites. In this work it can be seen how this chemical agent affects in a significant way the proximal tubule by increasing Oat5 urinary excretion. In addition, it is important to remark that the highest dose used in this study is very far from the highest dose applied in human chemotherapeutic regimens, pointing out the need for a diagnostic method to detect MTX-induced AKI at a time point when it is possible to take emergency measurements to prevent life-threatening toxicities.

Great efforts arise from the issue of MTX-plasma level monitorization with the application of different analytical, pharmacokinetic and mathematical methods to help predicting MTX toxic concentrations and diminish potential side effects. Even more, there are plenty of technical approaches, such as dialysis-based methods and glucarpidase administration, to improve MTX-removal. Nevertheless, protocols are controversial and, nowadays, there is no ideal one for these purposes. The present study contributes to further understanding MTX nephrotoxicity and enriches the possibility to find a noninvasive biomarker in order to detect alterations on MTX elimination, due to renal tubular damage. This disclosure has clinical relevance, not only to foresee MTX-induced AKI, but to anticipate an eventual systemic toxicity, due to its inherent pharmacokinetics. Oat5 would be useful as an early and novel biomarker of MTX-induced AKI, as it is altered before any conventional biomarker does. Then, Oat5 would have the ability to predict and, if it is possible, prevent the subsequent development of MTX-induced AKI.

AKI is associated with significant early and late mortality, even after 10 years and regardless of the degree of functional recovery. The incorporation of damage biomarkers into clinical practice awaits agreement regarding the appropriate thresholds (cut-offs) for diagnosis or categories of severity.32 Before a new biomarker can be deemed clinically useful, it requires validation in multiple cohorts, it must be reliable, and it must provide incremental prognostic information over traditional markers and models.33 Therefore, more studies are required to translate these results to clinics.

3. Experimental

3.1. Experimental animals

Male Wistar rats aged 110–130 days were used throughout the study. The animal protocol was designed to minimize pain or discomfort to the animals. Animals were cared for in accordance with the principles and guidelines for the care and use of laboratory animals, recommended by the National Academy of Sciences and published by the National Institute of Health (NIH publication 7th edition revised 1996) and recommended by regulations of the local ethics committee. All experimental procedures were approved by the Faculty of Biochemical and Pharmaceutical Sciences Institutional Animal Care and Use Committee (Res. N° 484/2015).

3.2. Experimental protocols

The animals were randomly divided into six experimental groups. Rats were treated with a single i.p. injection of MTX at different dosages: 40, 80, 120, 180 and 360 mg per kg b.w. (M40, M80, M120, M180, M360, n = 4, respectively). Control group (control, n = 4) received the vehicle only (1 mL saline per kg b.w., i.p.) and was treated simultaneously. The doses of MTX were chosen considering the application as a chemotherapeutic and anti-inflammatory agent in humans.5,34–37 After 24 h of the injection (saline or MTX), the animals were transferred to metabolic cages for urine collection for the next 24 h, without food to improve urine sample quality as described in Pinches et al.38 The urinary volume (VU) was determined gravimetrically. VU was expressed as mL per 24 h per 100 g b.w.

The studies were performed 48 h after the injection. On the day of the experiment, the rats were anesthetized with sodium thiopental (70 mg per kg b.w., i.p.) and plasma samples were obtained by cardiac puncture and kidneys were removed.

Two different sets of experimental animals were used: one for biochemical determinations and preparation of urine samples, total homogenates and apical membranes from kidney, and another for histopathological and immunohistochemistry studies.

3.3. Biochemical determinations

The urine samples were used for analyses of Oat5 abundance, alkaline phosphatase (AP) activity, creatinine (CrU) and proteins levels (PrU). Serum samples were used to measure urea (UrP) and creatinine levels (CrP). Serum urea and creatinine levels, as well as urine creatinine concentration, AP activity and PrU were determined spectrophotometrically employing commercial kits (Wiener Laboratory; Rosario, Argentina). Creatinine clearance (ClCr) was calculated by the conventional formula: ClCr = (CrU × VU)/CrP. Water fractional excretion (FEWater, %) was calculated by the formula FEWater = (VU/ClCr) × 100.

3.4. Preparation of total homogenates and apical membranes from kidneys

Apical membranes were isolated from kidneys by Mg/EGTA precipitation as previously described.17,18 Kidneys were removed, decapsulated, cleaned in cold saline, dried and weighed. The renal tissue was minced and homogenized in 30 g per 100 mL of ice-cold 50 mM mannitol, 5 mM EGTA, 10 mM HEPES-Tris HCl buffer (pH 7.40) and 1 mM phenylmethylsulfonyl fluoride (PMSF) for 5 min at top speed in a Glas-Col homogenizer. From this preparation, we obtained total renal homogenates, and aliquots were taken and stored at –80 °C until use. MgCl2 was added to the homogenate to a final concentration of 12 mM, and the mixture was stirred in an ice bath for 15 min. The homogenate was then centrifuged (3000g, 15 min, 4 °C). The pelleted material representing apical membranes was resuspended in 50 mM mannitol, 10 mM Hepes-Tris (pH 7.50) and 1 mM PMSF and centrifuged for 15 min at 800g at 4 °C. The supernatant was finally centrifuged for 45 min at 28 000g. The apical membrane pellets, thus obtained, were resuspended in 50 mM mannitol, 10 mM Hepes-Tris (pH 7.50) and 1 mM PMSF. Aliquots of the membranes were stored immediately at –80 °C for 2 weeks. Each preparation represented renal tissues from four animals. Protein quantification of samples was performed using the method of Lowry with some modifications.39

3.5. Electrophoresis and immunoblotting

Total renal homogenates (20 μg of protein), apical membranes (20 μg of protein) and urine (10 μL) samples were boiled for 3 min in the presence of 1% 2-mercaptoethanol and 2% sodium dodecyl sulphate (SDS). Samples were applied to an 8.5% polyacrylamide gel, separated by SDS-PAGE, and then electroblotted to nitrocellulose membranes (NC membrane). NC membranes were stained with Ponceau Red to confirm equal protein loading and transfer between lanes as previously described.40 The NC membranes were incubated with 5% non-fat dry milk in PBS containing 0.1% Tween 20 (PBST) for 1 h. After being rinsed with PBST, the membranes were incubated overnight at 4 °C with a non-commercial rabbit polyclonal antibody against rat Oat5 (at a dilution of 1 : 800). The specificity of Oat5 antibody has been described elsewhere.15 NC membranes were incubated for 1 h with a peroxidase coupled goat anti-rabbit IgG (Amersham, Buckinghamshire, UK) after further washing with PBST. Blots were processed for detection using a commercial kit (ECL enhanced chemiluminescence system, Amersham, Buckinghamshire, UK). A densitometric quantification of the western blot signal intensity of membranes was performed. For densitometry of immunoblots, samples from treated rats were run on each gel with corresponding control samples. The abundance of Oat5 in the samples from the experimental animals was calculated as percentage of the mean control value for that gel, and expressed as arbitrary units related to urinary creatinine concentration in order to correct for variations in urine production.

3.6. Histopathological and immunohistochemistry studies

A different set of experimental animals were used for histopathological and immunohistochemistry studies as previously described.17,18 Kidneys were briefly perfused with saline, followed by perfusion with periodate–lysine–paraformaldehyde solution (0.01 M NaIO4, 0.075 M lysine, 0.0375 M phosphate buffer, with 2% paraformaldehyde, pH 6.20) through a cannula introduced in the abdominal aorta. The kidney slices were immersed in periodate–lysine–paraformaldehyde solution at 4 °C overnight and embedded in paraffin. Then, 4 mm thick paraffin sections were cut. After deparaffinating, some sections were processed for routine staining with hematoxylin–eosin. Another set of sections was used for Oat5 immunohistochemistry and were incubated with 3% H2O2 for 15 min (to eliminate endogenous peroxidase activity) and then with blocking serum for 30 min. The sections were then incubated with a non-commercial polyclonal antibody against Oat5 (diluted 1 : 100) overnight at 4 °C. The sections were rinsed with PBST and then were incubated with horseradish peroxidase (HRP) conjugated secondary antibody against rabbit immunoglobulin for 1 h. In order to detect HRP labeling, a peroxidase substrate solution with diaminobenzidine (0.05% diaminobenzidine in PBST with 0.05% H2O2) was used. The sections were counterstained with hematoxylin before being examined under a light microscope.

3.7. Materials

Chemicals were purchased from Sigma (St. Louis, MO, USA) and were analytical grade pure. The rabbit polyclonal antibody against Oat5 was kindly provided by Prof. N. Anzai (Department of Pharmacology and Toxicology, Dokkyo Medical University School of Medicine, Tochigi, Japan.).

3.8. Statistical analysis

Statistical differences between groups were evaluated using the unpaired Student's t-test or multiple comparisons with one way ANOVA followed by the Newman–Keuls test. p values of less than 0.05 were considered statistically significant. The values are expressed as the means ± standard error (SEM). For these analyses, GraphPad 6 software (San Diego, California) was used.

4. Conclusions

Oat5 is found in urine at different MTX doses when no other alterations for kidney damage, such as traditional urine and blood markers, had been detected. Data from this work provide more information to propose Oat5 urinary excretion as a potential and noninvasive biomarker to an emerging biomarker panel to detect renal damage at a shorter time point when medical care might take action to prevent further complications.

Acknowledgments

This study was supported by the following grants: Fondo para la Investigación Científica y Tecnológica (FONCYT), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de Rosario (UNR). The authors thank to Prof. N. Anzai (Department of Pharmacology and Toxicology, Dokkyo Medical University School of Medicine, Tochigi, Japan.) for kindly providing Oat5-specific antibodies and Mrs Alejandra Martínez (Area Morfología, Facultad de Ciencias Bioquímicas y Farmacéuticas, U.N.R.) for her collaboration in the present work. The authors also thank Wiener Lab Argentina for analytical kits.

References

  1. Çaglar Y., Özgür H., Matur I., Yenilmez E. D., Tuli A., Gönlüs G., Polat S. Histol. Histopathol. 2013;28:865–874. doi: 10.14670/HH-28.865. [DOI] [PubMed] [Google Scholar]
  2. Tian H., Cronstein B. N. Bull. NYU Hosp. Jt. Dis. 2007;65:168–173. [PubMed] [Google Scholar]
  3. Green M. R., Chamberlain M. C. Cancer Chemother. Pharmacol. 2009;63:599–604. doi: 10.1007/s00280-008-0772-0. [DOI] [PubMed] [Google Scholar]
  4. Ackland S. P., Schilsky R. L. J. Clin. Oncol. 1987;5:2017–2031. doi: 10.1200/JCO.1987.5.12.2017. [DOI] [PubMed] [Google Scholar]
  5. Widemann B. C., Adamson P. C. Oncologist. 2006;11:694–703. doi: 10.1634/theoncologist.11-6-694. [DOI] [PubMed] [Google Scholar]
  6. Braun J. Ther. Adv. Musculoskeletal Dis. 2011;3:151–1586. doi: 10.1177/1759720X11408635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kuhn A., Ruland V., Patsinakidis N., Luger T. A. Clin. Exp. Rheumatol. 2010;28:S138–S144. [PubMed] [Google Scholar]
  8. Mallipattu S. K., Ross M. J. Kidney Int. 2011;80:226. doi: 10.1038/ki.2011.97. [DOI] [PubMed] [Google Scholar]
  9. Shirali A. C., Perazella M. A. Adv. Chronic Kidney Dis. 2014;21:56–63. doi: 10.1053/j.ackd.2013.06.010. [DOI] [PubMed] [Google Scholar]
  10. Kolli V. K., Abraham P., Isaac B., Selvakumar D. Chemoteraphy. 2009;55:83–90. doi: 10.1159/000192391. [DOI] [PubMed] [Google Scholar]
  11. Vaidya V. S., Ferguson M. A., Bonventre J. V. Annu. Rev. Pharmacol. Toxicol. 2008;48:463–493. doi: 10.1146/annurev.pharmtox.48.113006.094615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Peres L. A., Cunha Júnior A. D., Schäfer A. J., Silva A. L., Gaspar A. D., Scarpari D. F., Alves J. B., Girelli Neto R., Oliveira T. F. J. Bras. Nefrol. 2013;35:229–236. doi: 10.5935/0101-2800.20130036. [DOI] [PubMed] [Google Scholar]
  13. Ferguson M. A., Vaidya V. S., Bonventre J. V. Toxicology. 2008;245:182–193. doi: 10.1016/j.tox.2007.12.024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Youngblood G. L., Sweet D. H. Am. J. Physiol. 2004;287:F236–F244. doi: 10.1152/ajprenal.00012.2004. [DOI] [PubMed] [Google Scholar]
  15. Anzai N., Jutabha P., Enomoto A., Yokoyama H., Nonoguchi H., Hirata T., Shiraya K., He X., Cha S. H., Takeda M., Miyazaki H., Sakata T., Tomita K., Igarashi T., Kanai Y., Endou H. J. Pharmacol. Exp. Ther. 2005;315:534–544. doi: 10.1124/jpet.105.088583. [DOI] [PubMed] [Google Scholar]
  16. Di Giusto G., Anzai N., Endou H., Torres A. M. J. Histochem. Cytochem. 2009;57:17–27. doi: 10.1369/jhc.2008.951582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Di Giusto G., Torres A. M. Arch. Toxicol. 2010;84:741–749. doi: 10.1007/s00204-010-0541-9. [DOI] [PubMed] [Google Scholar]
  18. Bulacio R. P., Torres A. M. Arch. Toxicol. 2013;87:1953–1962. doi: 10.1007/s00204-013-1062-0. [DOI] [PubMed] [Google Scholar]
  19. Bulacio R. P., Torres A. M. Arch. Toxicol. 2015;89:1359–1369. doi: 10.1007/s00204-014-1345-0. [DOI] [PubMed] [Google Scholar]
  20. Hazelhoff M. H., Bulacio R. P., Torres A. M. BioMed. Res. Int. 2013;2013:283429. doi: 10.1155/2013/283429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Grönroos M., Chen M., Jahnukainen T., Capitanio A., Aizman R. I., Celsi G. Pediatr. Blood Cancer. 2006;46:624–629. doi: 10.1002/pbc.20471. [DOI] [PubMed] [Google Scholar]
  22. Bozkurt M., Em S., Oktayoglu P., Turkcu G., Yuksel H., Sariyildiz M. A., Caglayan M., Batmaz I., Nas K., Bozkurt Y., Kuyumcu M. Clin. Invest. Med. 2014;37:E19–E25. doi: 10.25011/cim.v37i1.20865. [DOI] [PubMed] [Google Scholar]
  23. Devarajan D., Murray P. Nephron. Clin. Pract. 2014;127:176–179. doi: 10.1159/000363206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Di Giusto G., Anzai N., Endou H., Torres A. M. Pharmacology. 2008;81:127–136. doi: 10.1159/000110555. [DOI] [PubMed] [Google Scholar]
  25. Van Vleet T., Schnellmann R. Semin. Nephrol. 2003;23:500–508. doi: 10.1016/s0270-9295(03)00094-9. [DOI] [PubMed] [Google Scholar]
  26. Uchino S., Kellum J. A., Bellomo R., Doig G. S., Morimatsu H., Morgera S., Schetz M., Tan I., Bouman C., Macedo E., Gibney N., Tolwani A., Ronco C. J. Am. Med. Assoc. 2005;294:813–818. doi: 10.1001/jama.294.7.813. [DOI] [PubMed] [Google Scholar]
  27. Lameire N., Van Biesen W., Vanholder R. Lancet. 2005;365:417–430. doi: 10.1016/S0140-6736(05)17831-3. [DOI] [PubMed] [Google Scholar]
  28. Reichel R. R. Ochsner J. 2014;14:359–368. [PMC free article] [PubMed] [Google Scholar]
  29. Visentin M., Zhao R., Goldman I. D. Hematol. Oncol. Clin. North. Am. 2012;26:629–648. doi: 10.1016/j.hoc.2012.02.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hagos Y., Wolff N. A. Toxins. 2010;2:2055–2082. doi: 10.3390/toxins2082055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Bonventre J. V., Vaidya V. S., Schmouder R., Feig P., Dieterle F. Nat. Biotechnol. 2010;28:436–440. doi: 10.1038/nbt0510-436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Endre Z. H. Biomarkers Med. 2014;8:1185–1188. doi: 10.2217/bmm.14.89. [DOI] [PubMed] [Google Scholar]
  33. Coca S. G. Kidney Int. 2008;73:1008–1016. doi: 10.1038/sj.ki.5002729. [DOI] [PubMed] [Google Scholar]
  34. Widemann B. C., Balis F. M., Kempf-Bielack B., Bielack S., Pratt C. B., Ferrari S., Bacci G., Craft A. W., Adamson P. C. Cancer. 2004;100:2222–2232. doi: 10.1002/cncr.20255. [DOI] [PubMed] [Google Scholar]
  35. Cronstein B. L. Pharmacol. Rev. 2005;57:163–172. doi: 10.1124/pr.57.2.3. [DOI] [PubMed] [Google Scholar]
  36. Braun J., Rau R. Curr. Opin. Rheumatol. 2009;21:216–223. doi: 10.1097/BOR.0b013e328329c79d. [DOI] [PubMed] [Google Scholar]
  37. Reagan-Shaw S., Nihal M., Ahmad N. FASEB J. 2007;22:659–661. doi: 10.1096/fj.07-9574LSF. [DOI] [PubMed] [Google Scholar]
  38. Pinches M. D., Betts C. J., Bickerton S. J., Beattie L., Burdett L. D., Thomas H. T., Derbyshire N. A., Moores M. Toxicol. Pathol. 2010;40:534–540. doi: 10.1177/0192623311432438. [DOI] [PubMed] [Google Scholar]
  39. Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. J. Biol. Chem. 1951;193:265–275. [PubMed] [Google Scholar]
  40. Brandoni A., Anzai N., Kanai Y., Endou H., Torres A. M. Biochim. Biophys. Acta. 2006;1762:673–682. doi: 10.1016/j.bbadis.2006.05.011. [DOI] [PubMed] [Google Scholar]

Articles from Toxicology Research are provided here courtesy of Oxford University Press

RESOURCES