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Available online at www.sciencedirect.com

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Carboplatin-induced Fanconi-like syndrome in rats: Amelioration by pentoxifylline Ravi Khakhariya a , S.P. Rathod a , Hardik Gandhi a,∗ , Bhavesh Variya a , Jinal Trivedi a , Prachi Bhamre b , S.J. Rajput b a

Pharmacology Laboratory, Pharmacy Department, Faculty of Technology and Engineering, The M.S. University of Baroda, India b Pharmaceutical Quality Assurance Laboratory, Pharmacy Department, Faculty of Technology and Engineering, The M.S. University of Baroda, India

a r t i c l e

i n f o

a b s t r a c t

Article history:

Introduction: Carboplatin is a congener of cisplatin used in the treatment of ovarian, head and

Received 22 July 2013

neck and small-cell lung cancer. However, the clinical efficacy of carboplatin is marred by the

Received in revised form

development of ROS-dependent nephrotoxicity. The pathophysiological damage inflicted

22 November 2013

upon the kidney by carboplatin closely resembles to that of Fanconi syndrome.

Accepted 27 November 2013

Aims and objectives: The present study aimed at inducing Fanconi-like syndrome in rats by

Available online 6 December 2013

administration of carboplatin. Objectives of the study involved evaluation of biochemical

Keywords:

the potential therapeutic effect of pentoxifylline in this condition.

parameters coherent to Fanconi-like syndrome. Further, an attempt was made to evaluate Carboplatin

Results: The results of the study demonstrated that the urinary excretion profile of car-

Cysteinuria

boplatin treated rats closely resembled to that of patients suffering from Fanconi-like

Fanconi syndrome

condition. Pentoxifylline was able to ameliorate this nephrotoxic condition as suggested

Pentoxifylline

by the change in levels of membrane bound ATPases, MDA and GSH. The urinary levels

Tyrosinuria

of tyrosine and cysteine correlate well with that of Fanconi-like condition in animals and humans. Conclusion: In lieu of these observations, our study suggested that carboplatin-induced renovascular damage resembles to Fanconi-like condition which can be mitigated by pentoxifylline. © 2013 Elsevier B.V. All rights reserved.

1.

Introduction

Carboplatin, a second-generation coordination complex of platinum (cis-diamine-1,1-cyclobutanedicarboxylateplatinum II), was approved for treatment of ovarian cancers in 1989. It is currently available clinically for the treatment of small cell lung cancer, ovarian and head and neck cancers (Bolis et al., 2001; Ettinger, 1998; Fujiwara et al.,

2003; Pivot et al., 2001). Carboplatin is more water soluble, more stable and less reactive but has comparable DNAdamaging activity to other platinum derivatives at equal doses (Alberts, 1995; McKeage, 1995; Meyer et al., 2001). In several chemotherapy regimens cisplatin is substituted by carboplatin as it has a milder adverse effect profile (English et al., 1999; McKeage, 1995) but clinically to achieve optimal results, dose of carboplatin has to be increased (Bohm et al., 1999; Wandt et al., 1999). The predominant dose-limiting

∗ Corresponding author at: Pharmacy Department, Faculty of Technology and Engineering, Kalabhavan, Dandia Bazaar, Vadodara 390001, Gujarat, India. Tel.: +91 265 2434187; fax: +91 265 2418927. E-mail address: [email protected] (H. Gandhi). 1382-6689/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2013.11.025

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toxicities of carboplatin include bone marrow suppression and ototoxicity (Cavaletti et al., 1998; Husain et al., 2001a; McKeage, 1995). In addition to this, platinum–amine–DNA adduct, a by-product of carboplatin metabolism, accumulates in the kidney and induces renal tubular damage (Kintzel, 2001). This toxic effect limits its application in cancer chemotherapy. Moreover, former reports (English et al., 1999; Yasumasu et al., 1992) shows that renal side effects are induced by carboplatin, especially when supplied in higher dosage regimens or in the presence of other risk factors like altered pretreatment GFR, cumulative dose of carboplatin or carboplatin dose intensity. Carboplatin is primarily excreted by the kidneys via tubular filtration and secretion; hence dose of carboplatin requires to be adjusted in patients with renal dysfunction (Agraharkar et al., 1998; English et al., 1999). The biochemical mechanism of nephrotoxicity induced by carboplatin has not been well understood. It has been hypothesized that carboplatin-induced renal injury is linked with enhanced free radical and reactive oxygen species generation as well as depletion of antioxidant molecules in the kidney (Husain et al., 2001a,b; Husain et al., 2004). Fanconi syndrome (also known as Lignac-de Toni-DebreFanconi syndrome) was first described in children with a clinical presentation of rickets, growth retardation, and glycosuria (Hou, 2009; Limsuwat and Prabhakar, 2012; Selvan et al., 2012). The renal Fanconi syndrome is an inherited or acquired metabolic disorder of proximal tubules characterized by tyrosinemia, cystinosis, Wilson’s disease, glycogen storage disease, galactosemia, and the oculo-cerebro-renal syndrome of Lowe (Foreman and Roth, 1989; Hurvitz et al., 1989). In addition to these, there is presence of various metabolic abnormalities like proteinuria, aminoaciduria, glucosuria, phosphaturia, bicarbonaturia, uricosuria and proximal renal tubular acidosis (Bokenkamp and Ludwig, 2011; Sirac et al., 2011). Fanconi syndrome is often observed in patients undergoing treatment with nephrotoxic drugs and other chemical agents. Exposure to some drugs and chemicals like outdated tetracyclines (Hawkins and Brewer, 1993), streptozotocin (Foreman and Roth, 1989), gentamicin (Melnick et al., 1994), cystine (Ben-Nun et al., 1993), valproic acid (Lande et al., 1993), 4-pentenoate (Pouliot et al., 1992), heavy metals (Fleck et al., 2001), ifosfamide (Burk et al., 1990; Mohrmann et al., 1993), maleic acid (Harrison and Harrison, 1954), and succinyl acetone (Wyss et al., 1992) lead to Fanconi-like condition. The characteristic feature of Fanconi syndrome is dysfunction of amino acid reabsorption which resembles the early phase of drug-induced nephrotoxicity (Burk et al., 1990). This fact has been exploited to utilize renal amino acid handling as a sensitive marker for drug-induced nephrotoxicity (Burk et al., 1990). Renal secretion of amino acids may thus be successfully used to monitor Fanconi syndrome. Pentoxifylline is a methyl-xanthine derivative that improves perfusion in the impaired microcirculation of peripheral and cerebral vascular beds (Ward and Clissold, 1987). This hemorheologic activity is due to inhibition of cyclic-3 ,5 -phosphodiesterase (PDE), which leads to raised intracellular cyclic adenosine monophosphate (cAMP) and activation of protein kinase A (PKA). Pentoxifylline inhibits PDE 1–5 with IC50 values ranging from 50 to 200 ␮M, hence it is classified as a non-selective PDE inhibitor (Meskini

et al., 1994). It has confirmed and potent inhibitory effects on cell proliferation, inflammation, and extracellular matrix accumulation. Pentoxifylline can also suppress activation and proliferation of mesangial cells, lymphocytes, and renal fibroblasts, all of which play significant roles in renal fibrosis (Chen et al., 1999; Lin et al., 2003; Tsai et al., 1995). It has been established that pentoxifylline can diminish proteinuria in diabetic patients (Navarro et al., 1999) attributed in part, to its hemorheologic action in addition to its anti-TNF-␣ action. All these reports suggested that pentoxifylline administration might find benefit in the condition of renal insults. In lieu of these reports, the present study was designed to evaluate the effect of pentoxifylline in carboplatin induced Fanconi-like syndrome.

2.

Materials and methods

2.1.

Animals

Adult, healthy albino rats of either sex (Wistar strain), weighing between 300 and 350 g were used for the study. Experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Pharmacy Department, Faculty of Tech. & Engg., The M.S. University of Baroda, Vadodara (vide Protocol no. MSU/PHARM/IAEC/2011/03). All experimental procedures were carried out in accordance to the CPCSEA guidelines. Animals were housed in an airconditioned room (25 ± 2 ◦ C, 30–65% RH) in polypropylene cages (not more than 3 animals per cage) having paddy husk (Shree Dutt Agro Pvt. Ltd., Vadodara) as bedding with 12 h light/dark cycles with free access to pelleted diet (Pranav Agro Foods Pvt. Ltd., Vadodara) and tap water.

2.2.

Drugs and reagents

Carboplatin was a generous gift from Sun Pharmaceuticals Pvt. Ltd., Mumbai). Pentoxifylline injection (TRENTAL® , SanofiAventis) was purchased from the local market. All other reagents and chemicals used were of analytical grade. Carboplatin was dissolved in 0.9% (w/v) NaCl to give a final concentration of 10 mg/ml and from this solution a single dose was administered to the animals. Pentoxifylline was injected as such from marketed formulation. All the solutions were freshly prepared prior to administration to animals.

2.3.

Experimental protocol

All the animals were divided into 4 different groups of 5 animals each. Group 1 (control): animals treated with equal volume of normal saline served as vehicle control (i.p.). Group 2 (carboplatin control): animals were given single dose of carboplatin (25 mg/kg, i.p.). Group 3 (carboplatin + pentoxifylline treated): after 1 h of carboplatin injection, animals were treated with pentoxifylline (45 mg/kg, i.p.). Group 4 (pentoxifylline control): animals were treated with single dose of pentoxifylline only.

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2.4.

Biochemical analyses

Urine samples were collected up to 24 h after administration of pentoxifylline and at the end of this period blood samples were collected from the retro orbital sinus under mild ether anesthesia. Animals were sacrificed after urine and blood collection, both kidneys were removed and stored in 10% buffered formalin for histopathological studies. For estimation of serum parameters, blood samples were allowed to stand undisturbed for 10 min at room temperature. They were then centrifuged at 1000 × g for 15 min to separate the serum. The supernatant was collected and used for estimation of blood urea nitrogen (BUN) (Patton and Crouch, 1977), serum creatinine (Bartels et al., 1972) and uric acid (Newman and Price, 1999) levels. Urine samples collected over time were used for the estimation of urinary flow rate (Qu ), albumin (McLauchlan, 1988), creatinine (Bartels et al., 1972), creatinine clearance (CrCl), total protein (Lowry et al., 1951), glucose (Sasaki and Sonnae, 1972), cysteine and tyrosine. Kidneys were homogenized in 100 mM tris–HCl buffer (pH 7.4) and centrifuged at 10,000 × g to separate the supernatant and the residue. The supernatant was assayed for reduced glutathione (GSH) and malondialdehyde (MDA) while membrane bound ATPase enzymes were estimated in the residue which was resuspended in 100 mM tris–HCl buffer. Protein content in this residual mixture was determined by the method described previously (Lowry et al., 1951).

2.5.

Assay of cysteine and tyrosine in urine

For estimation of individual amino acid in urine, a method reported by Suresh Babu et al. was followed. The assay involved amino acid profiling of more than 20 amino acids in urine using precolumn derivatization with OPA (orthopthalaldehyde) followed by reverse phase high performance liquid chromatography (RP-HPLC).

2.5.1.

Materials for HPLC

All the chemicals used in this method were of analytical grade. For mobile phase, methanol – solvent A (HPLC grade, Fischer Scientifics) and 0.05 M acetate buffer pH 6.8 – solvent B (prepared in double distilled water) were used as solvents. For preparation of samples; sodium borate buffer (0.5 M, pH 10.5): 7.7 g of boric acid dissolved in 200 ml distil water and then pH was adjusted to 10.5 using 2 N NaOH. OPA reagent: 10 mg of OPA (HiMedia, India) dissolved in 1 ml of methanol – sodium borate buffer (1:9, v/v) mixture containing 0.01% (v/v) ␤-mercaptoethanol added at the time of use (Loba Chemie, India). Alkylating reagent: 0.2 M iodoacetate (Sigma Chem. Co., USA) in sodium borate buffer. Standard cysteine and tyrosine were obtained from Loba Chemie, India. Methanol: water mixture (8:2, v/v) was used to make standard amino acid stock solutions and stored at 4 ◦ C until use.

2.5.2.

Table 1 – Solvent system for gradient analysis. Time (min) 0.01 5.00 10.00 25.00 35.00 40.00 45.00 47.00 50.00

Conc. of solvent A (%)

Conc. of solvent B (%)

90 80 74 64 25 25 50 90 Stop

10 20 26 36 75 75 50 10 Stop

allowed to stand for 5 min at room temperature. Mixture was precipitated using 395 ␮l of ice cold methanol using vortex mixer. Tubes were kept in ice bucket for 15 min and after that centrifuged at 1800 × g for 15 min. Bradford’s dye binding method was used for checking efficiency of protein precipitation (Bradford, 1976). The protein free supernatants were collected and used for HPLC. For precolumn derivatization 100 ␮l of urine extract was added into 50 ␮l sodium borate buffer containing iodoacetate (0.2 M) followed by OPA reagent (25 ␮l). 825 ␮l of start eluent (mixture of acetate buffer, 0.05 M, pH 6.8:methanol; 4:2, v/v) was added to that solution to make it up to 1 ml. Next, solution of amino acid derivatized with OPA was injected to HPLC system (Shimadzu, Japan). System was equipped with 20 ␮l injection loop and Phenomenex Luna analytical column (150 mm × 4.6 mm, 5 ␮m) with LC-20 AT pump fitted with a guard column (1 cm) which was kept in an incubator oven (Mayura Analytical Pvt. Ltd., Bangalore) set at 40 ◦ C constant temperature. All the amino acids were separated individually by reverse phase gradient of solvent A and solvent B as given in Table 1 at the flow rate of 100 ␮l/min. Fluorescence detector (Shimadzu RF-20A) was used to monitor the resolution of individual amino acids with excitation and emission set at 330 nm and 450 nm respectively. Calibration curve for both cysteine and tyrosine were prepared using the same procedure as described above. The regression equations (with R2 values approaching unity) obtained for cysteine y = 5137x − 27,222 and tyrosine y = 25,632x − 72,509 (where, y = AUC of tyrosine peak and x = concentration of cysteine/tyrosine in sample) were used for the purpose of quantification of the amino acids in test samples.

2.6.

Assay of MDA and GSH

MDA formation was estimated by the method reported earlier (Slater and Sawyer, 1971). The results were expressed in terms of nM of MDA/mg of protein. GSH was estimated by the method described previously (Moron et al., 1979). The results were expressed in terms of ␮g of GSH/mg of protein.

2.7.

Assay of membrane bound ATPases

2.7.1.

Assay of Na+ /K+ -ATPase activity

Procedure for amino acid analysis

Amino acids were analyzed by high-performance liquid chromatography. Urine samples were collected overnight using metabolic cage and they were centrifuged at 1000 × g for 15 min to clear it from cells or debris. After that 100 ␮l of supernatants were mixed with 5 ␮l of ␤-mercaptoethanol and

Na+ /K+ -ATPase activity was measured from the amount of Pi (inorganic phosphorus) released according to the method described formerly (Bonting, 1970). Briefly, the reaction mixture was prepared such that 2 ml of the final assay solution

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contained 92 mM tris-buffer, 5 mM MgSO4 , 60 mM NaCl, 5 mM KCl, 0.1 mM EDTA and 4.0 mM ATP. Solution was allowed to incubate at 37 ◦ C for 10 min. After incubation reaction was started by adding of 200 ␮l supernatant of kidney homogenate. Mixture was incubated for further 30 min and reaction was stopped by addition of 2 ml of ice cold 10% TCA. The resulting mixture was centrifuged at 1000 × g and the supernatant was used for the estimation of Pi . Earlier described method (Cyrus and Subbarow, 1925) was used for estimation of the liberated Pi . Unit of enzyme activity was ␮M of Pi liberated/mg protein/h.

2.7.2.

3.

Results

3.1.

Biochemical estimations

3.1.1.

Serum parameters

Assay of Mg++ -ATPase

Mg++ -ATPase was assayed by the method described earlier (Ohnishi et al., 1982). 100 ␮l of 375 mM tris–HCl buffer, pH 7.6, 100 ␮l of 205 mM MgCl2 , 100 ␮l of 10 mM ATP and 100 ␮l of distilled water were taken in one test tube. 100 ␮l supernatant of kidney homogenate was added in that mixture and allowed to incubate for 15 min at 37 ◦ C. Finally, the reaction was stopped by adding 100 ␮l of 10% TCA. Tubes were centrifuged at 1000 × g and enzyme activity was expressed as ␮ moles of Pi liberated/mg protein/min.

2.7.3.

in test tubes. 100 ␮l supernatant of kidney homogenate was added and the tubes were incubated at 37 ◦ C for 15 min. The reaction was arrested by the addition of equal volume of 10% TCA to the incubation mixture. All the tubes were centrifuged at 1800 × g for 5 min and supernatant was used for the estimation of Pi . Unit of enzyme activity was ␮moles of Pi liberated/mg protein/min.

Assay of Ca++ -ATPase

Ca++ -ATPase was assayed by the method described previously (Hjerten and Pan, 1983) was used for the assay of Ca++ -ATPase. 125 mM tris–HCl buffer, pH 8.0, 100 ␮l 50 mM CaCl2 , 100 ␮l 10 mM ATP solution and 100 ␮l distilled water were taken

Carboplatin intoxication resulted in a two-fold increase in BUN levels (Fig. 1a). Treatment with PTX significantly reduced the elevated levels of BUN suggesting a protective effect. Serum creatinine levels were increased in carboplatin-treated animals suggesting a marred renal physiology (Fig. 1b). Administration of PTX was able to moderate these elevations in serum creatinine levels. Serum from animals treated with carboplatin showed dwindling levels of uric acid (P < 0.01) which was improved with PTX treatment (Fig. 1c). The results suggested that, PTX, by virtue of its PDE-inhibition ability or some other unknown mechanism, resolved the levels of markers of glomerular filtration in carboplatin-intoxicated rats.

Fig. 1 – Effect of carboplatin intoxication and PTX treatment on: (a) blood urea nitrogen (BUN), (b) serum creatinine, (c) serum uric acid and (d) renal GSH levels. PTX affected only a moderate rise in GSH levels after carboplatin administration. PTX did not mediate any untoward changes in these parameters. ***P < 0.001, **P < 0.01, *P < 0.05 and ‘ns’, non-significant difference between groups. For all experiments n = 5.

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Fig. 2 – Effect of carboplatin intoxication and PTX treatment on: (a) tissue lipid peroxidation, (b) urinary flow rate (Qu ), (c) urinary glucose excretion and (d) creatinine clearance. Lipid peroxidation expressed as nM of MDA released in the tissue. Urinary flow rate was normalized in terms of body-weight. ***P < 0.001, **P < 0.01, *P < 0.05 as indicated between different groups. For all experiments n = 5.

3.1.2.

Tissue parameters

Oxidative stress plays a major role in induction of nephrotoxicity by chemical agents like carboplatin. To evaluate the effect of carboplatin administration on the kidney, GSH and MDA levels were estimated in kidney homogenates of all the groups (Figs. 1d and 2a). GSH, a scavenger of free radicals, was found to be depleted in animals administered with carboplatin indicating the role of ROS and diminishing antioxidant defenses in nephrotoxic conditions. PTX mediated improvements in renal GSH levels were only marginal in the carboplatin-intoxicated group since the change in GSH levels is very small and this is contrary to previous reports (Seifi et al., 2012) involving PTX in renal conditions. However, it was presumed that this might be due the extremely short protocol used for PTX treatment. TBARS, quantified by the levels of malondialdehyde, are products of lipid peroxidation which were increased

up to 10-fold in carboplatin intoxicated group. In contrast to GSH, MDA levels were significantly reduced by PTX in both the groups. The effects of nephrotoxic insults on the membrane-bound ATPases are summarized in Table 2. The data from intoxicated animals indicated a significant derangement in the function of these enzymes as compared to the control animals. Activity of all the three studied enzymes expressed as a function of inorganic phosphate released, were reduced in the carboplatin-intoxicated animals. PTX was able to improve the activity of all the membrane-bound enzymes (Table 2).

3.1.3.

Urine parameters

Urinary flow rate is the first and foremost parameter to be altered in case of any type of renal dysfunction irrespective of the disease pathophysiology. In the present study, carboplatin

Table 2 – Effect of different treatments on ATPases.a ATPases +

+

Na /K ATPase Mg++ -ATPase Ca++ -ATPase a ∗∗ ∗ #

Group I

Group II

Group III

374.37 ± 2.97 301.66 ± 4.61 306.13 ± 2.71

268.28 ± 1.28 203.72 ± 2.56** 201.49 ± 1.85** **

Group IV

320.11 ± 11.37 248.28 ± 2.07* 258.77 ± 5.09#

All values are indicated as mean ± SEM (n = 5). Unit, ␮mol of inorganic phosphate liberated/mg of protein. P < 0.001 as compared to Group I. P < 0.01 as compared to Group II. P < 0.001 as compared to Group II.

*

369.81 ± 2.18 302.07 ± 4.07 299.98 ± 2.42

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Fig. 3 – Changes in urinary protein and amino acid excretion patterns following carboplatin intoxication were observed as: (a) proteinuria in terms of total protein spilled in the urine, (b) cysteinuria and (c) tyrosinuria. Cysteine levels rose 3-fold following carboplatin intoxication while tyrosine levels rose 3.5-fold. ***P < 0.001 as compared to Group I (control) and Group II (carboplatin).

increased the per minute urinary flow rate (Qu ). Carboplatin administration led to a 4- to 5-fold increase in urinary flow rate suggesting a decrease in urine concentrating ability of the kidney (Fig. 2b). Pentoxifylline treated animals showed a near-normal urinary flow rate. Glucosuria was another prominent observation in the study (Fig. 2c), with total urine output in carboplatin intoxicated animals being twice as that of normal. Pentoxifylline normalizes this effect suggesting that pentoxifylline interferes with the nephrotoxic potential of carboplatin. One of the most significant parameters denoting efficiency of renal clearance ability is creatinine clearance. This parameter was decreased in carboplatin intoxicated rats which is in corroboration with urine and serum creatinine levels (Fig. 2d). PTX treatment showed a modest improvement in creatinine clearance levels. PTX, per se, does not interfere with renal clearance of creatinine. Albumin is a macromolecular globular protein which is filtered by the kidney. Increased albumin secretion in the urine is suggestive of renal parenchymal damage and decreased ability of the nephrons to retain macromolecules. Albumin and simultaneously total protein output was increased in carboplatin intoxicated rats (Fig. 3a). Pentoxifylline was able to control protein spillage in the urine and per se did not have any effect on urinary protein output. These results also prompted the evaluation of renal amino acid handling. Since Fanconi-like condition is associated with decreased reabsorption of amino acids like cysteine and tyrosine (Roth et al., 1989) we evaluated all the groups for cysteinuria and tyrosinuria. It was observed that cysteine and tyrosine excretion in the urine increased by 3-fold (Fig. 3b) and 3.5-fold (Fig. 3c) respectively in carboplatin-intoxicated animals as compared to control ones. This finding was most suggestive for Fanconi-like condition and PTX administration normalized this aminoaciduria.

4.

Discussion

Carboplatin, a second generation platinum derivative is widely employed in the chemotherapy of lung, ovarian, head and neck cancers. Carboplatin has a significant advantage over cisplatin in terms of its nephrotoxicity being significantly

lower compared to cisplatin. It can therefore be given in higher doses to achieve optimal anti-neoplastic action. However, one of the metabolic byproducts of carboplatin, a platinum–ammine–DNA adduct has the tendency to accumulate in the kidney and induce damage to the renal tubules. This is one of the dose-limiting adverse effects related to clinical use of carboplatin. Several studies have shown that carboplatin administration can lead to dose-dependent nephrotoxicity in rodents and human beings specifically leading to acute tubular necrosis (Haschke et al., 2010; Husain et al., 2002, 2004). We have tried to corroborate these findings in rats treated with an overdose of carboplatin and tried to correlate carboplatin-induced renal damage to that of impairment of renal function observed in experimentally induced Fanconilike syndrome. BUN, creatinine and uric acid are the biomarkers of glomerular filtration. BUN level provides a generalized information regarding overall renal excretory function. Increase in BUN levels is indicative of diminishing ability of the kidneys to maintain the excretory profile. BUN levels are inversely proportional to GFR values and in the present study we found elevated BUN levels in animals intoxicated with carboplatin suggesting a decline in GFR in those animals. Other studies have also shown that carboplatin induces an increment in the BUN levels (Chen et al., 2010). A similar observation was also found for serum creatinine levels which are indicative of renal filtering ability (Chen et al., 2010). It has been also shown that carboplatin induced renal damage can lead to reduced excretion of serum uric acid as a factor of declining renal function (Kintzel, 2001). All these parameters collectively imply toward an unfavorable alteration of renal function. This study indicated that carboplatin intoxication can result in remarkable oxidative changes to the kidney as suggested by MDA and GSH levels in kidney homogenates. This is in relation to the profile of Fanconi-like syndrome in rats (Chen et al., 2008). Depletion of the antioxidant GSH allows lipid peroxidation to occur and this oxidative stress leads to the loss of functional integrity of the cell. Husain et al. concluded that carboplatin administration to rats can lead to increased lipid peroxidation and decreased levels of reduced glutathione. This statement has been corroborated by the works of Nowak

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and Janczak (2006) and Hannemann et al. (1991). In accordance with these findings, the present showed that carboplatin leads to an overall gloomy pro-oxidant profile in the kidney. These findings illustrate that in parallel to elevations in serum urea nitrogen and serum creatinine levels there is a concomitant rise in oxidative stress in the renal capsule. Reabsorption of water and other solutes like amino acids, phosphates, bicarbonates and glucose mainly occurs through the PCT (Berry and Rector, 1991). Specifically, this transport is effected by Na+ -coupled mechanisms involving the Na+ /K+ ATPase which maintains the low levels of intracellular low Na+ levels essential for successful Na+ -coupled (Eiam-ong et al., 1995). Decreased activity of the Na+ /K+ -ATPase leads to impaired membrane recycling activity and this was also observed in our model with carboplatin intoxication. As it is evident from Table 2, renal Na+ /K+ -ATPase activity was significantly reduced which stands in rank with other studies suggesting that renal insult lead to reduced Na+ /K+ -ATPase activity (Eiam-ong et al., 1995; Thevenod and Friedmann, 1999). We observed similar findings with the activity of other ATPases. We evaluated a battery of urinary parameters in carboplatin-intoxicated rats and found that the values found in these animals closely resembles to Fanconi-like condition. Evidently it was seen from the urine collection that the total urine output had increased in carboplatin intoxicated animals. This observation is common to several conditions related to renal dysfunction like TIN, diabetic nephropathy and the likes. Additionally, as mentioned earlier, increased BUN levels and increased urine output testify toward a reduced GFR in carboplatin-administered animals. The significant rise in the total urine output is suggestive of the loss of the urine concentrating ability of the kidney and represents the loss of nutrients like glucose, amino acids and minerals like bicarbonate and phosphate. This was again confirmed with our findings on glucosuria and proteinuria. Glucose is normally reabsorbed from the kidney and negligible amounts appear in the urine whereas proteins like albumin are macromolecules and hence are readily filtered at the glomeruli. In line with other reports (Chen et al., 2008; Sener et al., 2004), we found a significant excretion of glucose and total protein in the animals that were administered carboplatin. This suggested a loss of glomerular retention and reabsorptive capacity of the kidneys. We could thus suppose a major compromise of renal function upon carboplatin administration. To corroborate these findings creatinine clearance was evaluated in all the groups of animals and found expectedly that creatinine clearance had decreased in carboplatin intoxicated animals. These results in totality speak about a kidney approaching the condition of one with Fanconi syndrome. One of the major finding in urine of patients suffering from Fanconi syndrome (Long et al., 1990) and in experimental studies (Gunther et al., 1979) is aminoaciduria. This condition particularly refers to the spillage of different amino acids in urine, which, in the case of Fanconi syndrome are mostly cysteine (Walsh and Unwin, 2012) and tyrosine (Roth et al., 1989). This finding has been reported in several experimental studies involving Fanconi syndrome induced by either maleate (Nissim and Weinberg, 1996) or ifosfamide (Mohrmann et al., 1993). An ingenuous

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method (Babu et al., 2002) was used for the identification of cysteine and tyrosine in the urine of carboplatin treated animals. Results showed amplified cysteinuria and tyrosinuria affording credence to our speculation that carboplatin might induce a Fanconi-like condition in rats. With all these results at hand it was confirmed that carboplatin administration induces Fanconi-like condition in rats. The terminal part of our study involved evaluation of the effect of acute PTX administration to carboplatin-intoxicated rats as a measure of its ability to provide relief from the symptoms of Fanconi-like syndrome. PTX was able to ameliorate the marred biochemical profile of carboplatin intoxicated animals. Several studies have highlighted that PTX plays a nephroprotective role against unfavorable alteration of renal biochemical profile. PTX has also been studied for its potential role in diabetic kidney disease and alleviation of proteinuria in humans. In the present study, PTX did not show any major therapeutic benefit when the levels of antioxidant GSH were studied. We found that PTX only marginally affected the levels of GSH. This might be due to the acute mode of treatment involved in our protocol of study. However, PTX treated animals did show reduced levels of lipid peroxidation byproducts. This suggested that PTX displays some antioxidant action in this model which is unrelated to GSH level improvement. Mechanistic pathways by which pentoxifylline might be causing these favorable effects have not been evaluated in the present study. PTX has been previously employed in the experimental management of cyclosporine-nephropathy and mesangial proliferative glomerulonephritis (Albornoz et al., 1997; Chen et al., 1999). However, it can be observed upon retrospection that these effects might be due to PTX-mediated increase in renal blood flow through its hemorheologic action which is mediated through non-specific phosphodiesterase inhibition. Additionally, the renoprotective ability of PTX and its anti-TNF␣ action might also play a positive role in this protective effect (Ducloux et al., 2001; Navarro et al., 1999). It also has anti-fibrotic actions which are mediated through its ability to reduce lymphocyte profliferation and production of extracellular matrix proteins (Strutz et al., 2000). All these mechanisms might be acting in concert to afford relief from symptoms of renal dysfunction.

5.

Conclusion

It was observed that the biochemical changes associated with Fanconi syndrome are satisfactorily produced with carboplatin administration and this condition can be reasonably ameliorated with pentoxifylline, a non-specific PDE inhibitor. Herewith it is proposed that carboplatin-induced nephrotoxicity in rats closely resembles the biochemical profile of Fanconi-like condition. Clinically used PTX shows very few side effects and hence it has the potential to become a candidate for the treatment of Fanconi-like condition in humans. However, further work in relation to the present study should involve comparison of carboplatin-induced nephrotoxicity with Fanconi-like condition induced by other standard models like maleic acid- or ifosfamide-induced nephrotoxicity and the elucidation of mechanisms relevant to it.

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Conflict of interest The authors state that there are no conflicts of interest pertaining to this manuscript.

Acknowledgement The authors acknowledge the funding support provided to R.K. in the form of Post-graduate contingency by The M.S. University of Baroda.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.etap.2013.11.025.

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