Effect of uranyl nitrate on enzymes of carbohydrate metabolism and brush border membrane in different kidney tissues

Available online at www.sciencedirect.com

Food and Chemical Toxicology 46 (2008) 2080–2088 www.elsevier.com/locate/foodchemtox

Effect of uranyl nitrate on enzymes of carbohydrate metabolism and brush border membrane in different kidney tissues Anees A. Banday, Shubha Priyamvada, Neelam Farooq, Ahad Noor Khan Yusufi *, Farah Khan Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202 002, UP, India Received 15 January 2007; accepted 31 January 2008

Abstract Uranium, the heaviest of the naturally occurring elements is widely present as environmental contaminant from natural deposits, industrial emissions and most importantly from modern weapons. Histopathological examinations revealed that uranyl nitrate (UN) exposure caused severe damage to pars recta of renal proximal tubule. However, biochemical events involved in cellular response to renal injury are not completely elucidated. We hypothesized that UN exposure would severely damage kidney tissues and alter their metabolic functions. Rats were treated with a single nephrotoxic dose of UN (0.5 mg/kg body weight) i.p. After 5 d, effect of UN was studied on the activities of various enzymes of carbohydrate metabolism, brush border membrane (BBM) and oxidative stress in different kidney tissues. Activity of lactate dehydrogenase increased whereas activities of isocitrate, succinate and malate dehydrogenases, glucose-6-phosphatase and fructose-1,6-bisphosphatase significantly decreased by UN exposure. Activity of glucose-6-phosphate dehydrogenase decreased whereas that of NADP-malic enzyme increased. The activities of BBM enzymes were significantly lowered and after dissociation from BBM excreted in urine. Lipid peroxidation and the activities of superoxide dismutase and glutathione peroxidase increased whereas catalase activity decreased by UN. UN treatment caused specific alterations in the activities of metabolic and membrane enzymes and perturbed antioxidant defenses. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Uranyl nitrate; Nephrotoxicity; Brush border membrane enzymes; Carbohydrate metabolism; Oxidative stress; Rat kidney

1. Introduction The kidneys play an essential role in the maintenance of total body fluid volume, its composition and acid–base balance by selective reabsorption. A number of environmental Abbreviations: AcidPase, acid phosphatase; AlkPase, alkaline phosphatase; G6Pase, glucose-6-phosphatase; G6PDH, glucose-6-phosphate dehydrogenase; GGTase, c-glutamyl transferase; LAP, leucine aminopeptidase; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; SOD, superoxide dismutase; TCA cycle, tricarboxylic acid cycle; HMP, hexose monophosphate shunt; BBM, brush border membrane; BLM, basolateral membrane; BBMV, brush border membrane vesicles; JMCH, juxtamedullary cortex; SCH, superficial cortex; WCH, whole cortex; BUN, blood urea nitrogen; UN, uranyl nitrate. * Corresponding author. Tel.: +91 571 2700741; fax: +91 571 2706002. E-mail address: yusufi@lycos.com (A.N.K. Yusufi). 0278-6915/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2008.01.048

contaminants, drugs and renal ischemia dramatically alter these functions and produce multiple adverse effects (Cronin et al., 1986; Khundmiri et al., 1997; Goldman et al., 2006). The heavy metals such as mercury, platinum, chromium, lead and uranium have been shown to cause severe damage to kidney especially its proximal tubule (Cronin and Thompson, 1991; Domingo, 1994; Stochs et al., 2000; Sanchez et al., 2001; Fatima et al., 2004; Barbier et al., 2005). Uranium, the heaviest of the naturally occurring elements, is widely present in the environment as a result of leaching from natural deposits, industrial emissions and weapons that contain uranium. It is found in various chemical forms at different levels in all soils, rocks, sea and bedrocks (Bosshard et al., 1992; Kurttio et al., 2002). It is also found in both food and drinking water and pose significant health concern in several areas of North America

A.A. Banday et al. / Food and Chemical Toxicology 46 (2008) 2080–2088

(McDonald-Taylor et al., 1997). It is believed that the major health effect of uranium is chemical kidney toxicity rather than a radiation hazard (Miller et al., 1998, 2002). Uranyl nitrate induced nephropathy has been extensively studied in animal models (Haley et al., 1982; Anthony et al., 1994; Gilmen et al., 1998; Sanchez et al., 2001). Histopathological studies have shown that UN caused specific damage to pars recta (S2 and S3 subsegments) of renal proximal tubules (Haley et al., 1982). Some of the known effects include increased lysosomal and vacuolar mass; variations in mitochondrial mass; epithelial cell degeneration with a focal loss of brush borders, thickening and splitting of basolateral membrane (BLM) and occasionally cell necrosis (McDonald-Taylor et al., 1997; Gilmen et al., 1998). However, biochemical events and the mechanism involved in the cellular response to renal injury are not completely elucidated, neither those participating in inflammation, necrosis and oxidative stress or energy yielding metabolic activities. Recently we have shown that administration of cis-platinum (Fatima et al., 2004) and potassium dichromate (Fatima et al., 2005) caused injury to renal proximal tubules and resulted in decreased activities of BBM marker enzymes and Pi reabsorption similar to renal ischemia (Khundmiri et al., 1997, 2005). We now hypothesized that uranium exposure would severely damage kidney tissues and alter their metabolic functions. The primary aim of present work was to investigate the effect of administration of a single intraperitoneal dose of uranyl nitrate (UN) on the activities of various enzymes involved in carbohydrate metabolism, brush border membranes and antioxidant defenses in search of true markers of UN induced nephrotoxicity. The analysis was made in different renal tissue regions: e.g., whole, superficial and juxtamedullary cortices and medulla to identify the site of UN action. We observed that UN caused specific alterations in the activities of renal metabolic and membrane enzymes and perturbed antioxidant defense system; differentially in superficial and juxtamedullary cortical regions. 2. Materials and methods

2081

saline. Body weights of rats were recorded at start and completion of the experiment. Blood samples and specimens were collected before and after UN treatment. The rats were sacrificed under light ether anaesthesia 5 days after UN administration.

2.3. Preparation of homogenates to determine enzymes of carbohydrate metabolism After the completion of the experiment, the kidneys were removed, decapsulated and kept in ice-cold buffered saline (154 mM NaCl, 5 mM Tris–Hepes, pH 7.5). The whole (WCH), superficial (SCH) and juxtamedullary (JMCH) cortex was carefully separated from the medulla as previously described by Yusufi et al. (1994). A 15% (w/v) homogenate was prepared in 0.1 M Tris–HCl buffer pH 7.5. The homogenate was centrifuged at 800g at 4 °C for 10 min to remove cell debris. Aliquots of the homogenates were saved and stored at 20 °C until enzyme analyses.

2.4. Preparation of brush border membrane Brush border membrane vesicles (BBMV) were prepared from whole (WCH) superficial (SCH) and juxtamedullary (JMCH) cortex using the MgCl2 precipitation method as described previously (Khundmiri et al., 2005). Briefly, freshly minced cortical slices were homogenized in 50 mM mannitol and 5 mM Tris–Hepes buffer pH 7.0 (20 ml/g), in a glass Teflon homogenizer with 4 complete strokes. The homogenate was then subjected to high speed Ultra-Turrex Kunkel homogenizer (Type T-25, Janke & Kunkel GMBH & Co. KG. Staufen) for 3 strokes of 15 s each with an interval of 15 s between each stroke. MgCl2was added to the homogenate to a final concentration of 10 mM and the mixture stirred for 20 min on ice. The homogenate was centrifuged at 2000g in a J2-21 Beckman centrifuge (J2 MI, Beckman instruments. Inc. Palo Alto, CA, USA) and the supernatant was then recentrifuged at 35,000g for 30 min. The pellet was resuspended in 300 mM mannitol and 5 mM Tris–Hepes, pH 7.4, with 4 passes by a loose fitting Dounce homogenizer and centrifuged at 35,000g for 20 min in a 15 ml corex tube (Wheaton IL, Thomas PA, USA). The outer white fluffy pellet of BBM was resuspended in small volume of buffered 300 mM mannitol. Aliquots of homogenates and BBM were saved and stored at 20 °C until further use.

2.5. Serum and urine parameters They were analyzed as previously described (Khundmiri et al., 1997). Urinary enzymes were determined by standard colorimetric methods as described elsewhere. Na+ and K+ levels in urine were measured with flame fluorimeter (FLM-3, Denmark), and Ca2+ and Mg2+ were determined by using atomic absorbance spectrophotometer (Hitachi Z-7000, Japan) as described by Zhong et al. (1990).

2.1. Methods 2.6. Enzyme assays Uranyl nitrate was purchased from Sisco Research Laboratory (SRL), Mumbai, India. All other chemicals used were of analytical grade and were purchased either from Sigma Chemical Co. (St. Louis, MO, USA) or SRL, Mumbai, India.

2.2. Animal protocol The animal experiments were conducted according to the guidelines of Committee for Purpose of Control and Supervision of Experiments on animals, Ministry of Environment and Forests, Government of India. Adult male Wistar rats, weighing between 180 and 200 g, were acclimatized to the animal facility for a week on standard rat chow (Amrut Laboratories, Pune, India) and allowed water ad libitum. A single nephrotoxic dose of uranyl nitrate (0.5 mg/kg body wt) in saline was administered i.p. whereas control rats received an equivalent volume of normal

The activities of BBM enzymes, basolateral membrane enzymes, lysosomal enzymes and the enzymes of carbohydrate metabolism were determined on spectrophotometer (Cintra 5; GBC Scientific Equipment Pty, Victoria, Australia) by standard methods as described in previous studies (Khundmiri et al., 1997, 2004; Farooq et al., 2006).

2.7. Assay of enzymatic and non-enzymatic antioxidants Superoxide dismutase (SOD, E.C.1.15.1.1) was assayed by the method of Marklund and Marklund (1974). Catalase (E.C.1.11.1.6) and glutathione peroxidase (E.C.1.11.1.9) activities were determined by the method of Giri et al. (1996) and Flohe and Gunzler (1984), respectively. Total-SH was determined by the method of Sedlak and Lindsay (1968) and lipid peroxidation by the method of Ohkawa et al. (1979).

2082

A.A. Banday et al. / Food and Chemical Toxicology 46 (2008) 2080–2088

2.8. Statistical analysis All data are expressed as mean ± SEM for at least 5 separate experiments. Statistical evaluation was conducted by one-way ANOVA and/or by the student’s t-test. A probability level of p < 0.05 was selected as indicating statistical significance.

3. Results 3.1. Effect of UN on certain biochemical parameters of serum and urine The effect of a single nephrotoxic dose of UN was studied on various serum and urine biochemical parameters. In general, UN treatment did not change body weight and daily food intake of rats. Urine flow rate in UN treated rats rose nearly two fold thus indicating polyuria. Glucosuria was observed along with significant increase in electrolyte such as Na+, K+, Mg2+, Ca2+, Pi wasting and protein excretion (Tables 1A and 1B). However, UN treatment resulted in significant increase of serum creatinine (+112%), Blood Urea Nitrogen (BUN, +111%), cholesterol (+28%), and phospholipids (+100%) with concomitant decrease in serum Pi ( 31%) and creatinine clearance ( 61%) (Table 2A). In addition, several cellular enzymes of clinical importance e.g., alkaline phosphatase (AlkPase), c-glutamyl transpeptidase (GGTase), acid phosphatase (AcidPase) and lactate dehydrogenase (LDH) were profoundly excreted in the urine indicating a severe nephrotoxic effect of UN treatment (Table 2B). 3.2. Effect of UN on biomarkers of BBM in different tissue zones and isolated BBMV The effect of UN was determined on BBM marker enzymes and enzymes of other organelles/membranes. Relative enrichment of the BBM preparation was documented enzymatically using the apical membrane markers AlkPase,

GGTase, leucine aminopeptidase (LAP) and the basolateral membrane (BLM) marker Na+–K+ATPase. The results indicate that BBMVs prepared from WC, SC and JMC showed several fold enrichment (9–11-fold) of BBM enzymes as the activities of Na+–K+ ATPase (BLM), AcidPase (lysosomal) and succinate dehydrogenase (SDH) (mitochondrial) were not enriched as compared to respective homogenates (data not shown) demonstrating relative purity of BBMVs. The specific activities of AlkPase, GGTase, Maltase and LAP compared to control rats were significantly but differentially decreased in BBMV–WC, BBMV–SC and BBMV–JMC by UN treatment (Table 3). The activities of all the enzymes decreased to a greater extent in BBMV–JMC than BBMV–SC. Further, UN treatment caused a much greater effect on the activities of GGTase and LAP than on the activities of AlkPase and Maltase (Table 3). The decrease in the activities of the enzymes in BBMVs from UN treated rats relative to control was manifested by kinetic features of the enzymes. Apparent Km and Vmax values were determined graphically from double reciprocal plots (not shown). The data summarized in Table 4, show that Vmax (expressed as lmoles/ mg protein/h) of BBM enzymes was significantly lowered in UN treated rats as compared to the respective control values whereas apparent Km remained unchanged. The activities of AlkPase, GGTase, Maltase and LAP, however, were not lowered significantly by UN treatment in respective cortical homogenates (Table 3). The activities (expressed as total enzyme units) of the enzymes were further analyzed as membrane bound and free soluble enzyme by centrifugal separation. The results shown in Fig. 1 explicitly indicate that UN treatment caused decrease in membrane bound fractions whereas the activities of respective enzymes were found to be increased in free soluble fractions. This shows that UN treatment caused severe damage to brush border membrane due to which enzymes were dissociated and released from the membranes and appeared in free soluble form.

Table 1A Effect of UN treatment on body weight, food intake, and urine flow rate, urinary glucose and protein in intact animals Group

Body wt (g)

Food intake (g/day)

Urine flow rate (ml/day)

Protein (mg/mmole creatinine)

Glucose (mg/mg creatinine)

Control UN

200 ± 10 205 ± 10

20 ± 1 20 ± 1

15.4 ± 0.6 32.5 ± 0.8*

2.28 ± 0.10 16.30 ± 1.1*

15.0 ± 2.0 47.5 ± 2.5*

Results are mean ± SEM of five different experiments. * Significantly different at p < 0.05 as compared to saline-treated control.

Table 1B Effect of UN treatment on ion excretion in intact animals Group

Sodium

Potassium

Calcium

Magnesium

Inorganic phosphate

Control UN

3.0 ± 0.01 5.2 ± 0.019*

6.1 ± 0.18 8.8 ± 0.22*

3.0 ± 0.1 4.2 ± 0.08*

1.2 ± 0.01 1.5 ± 0.014*

1.6 ± 0.03 2.2 ± 0.06*

Results are mean ± SEM of five different experiments. Excretion of ions is expressed in meq/kg body wt/day and that of inorganic phosphate is expressed in lmoles/kg body wt/day. * Significantly different at p < 0.05 as compared to saline-treated control.

A.A. Banday et al. / Food and Chemical Toxicology 46 (2008) 2080–2088

2083

Table 2 Effect of UN treatment on (A) serum parameters (B) urinary enzymes Group (A) Control UN

Creatinine (mg/dl)

Creatinine clearance (ml/min/100 g body wt)

BUN (mg/dl)

Cholesterol (mg/dl)

Phospholipid (mg/dl)

Phosphate (lmole/ml)

0.42 ± 0.01 0.89 ± 0.07* (+112%)

0.33 ± 0.01 0.13 ± 0.01* ( 61%)

21.3 ± 1.0 45 ± 2.0* (+111%)

54.9 ± 3.0 70.3 ± 3.8* (+28%)

0.6 ± 0.02 1.2 ± 0.04* (+100%)

2.4 ± 0.11 1.65 ± 0.10 ( 31%)

Group (B) Control UN

GGTase

AlkPase

AcidPase

LDH

22.53 ± 1.0 80.0 ± 3.8* (+255%)

9.55 ± 1.0 14.7 ± 1.2* (+54%)

3.35 ± 0.2 14.1 ± 0.6* (+321%)

1.46 ± 0.03 4.2 ± 0.2* (+188%)

Results are mean ± SEM of five different experiments. Enzyme activities are expressed as units/mmole creatinine. Values in parenthesis represent percent change from respective control. * Significantly different at p < 0.05 as compared to saline-treated control.

Table 3 Effect of UN treatment on the activities of AlkPase, GGTase, maltase, LAP in homogenates and BBMVs isolated from whole cortex (WC), superficial cortex (SC) and juxtamedullary cortex (JMC) Group AlkPase Control UN GGTase Control UN Maltase Control UN LAP Control UN

WCH

BBMV–WC

SCH

BBMV–SCH

JMCH

BBMV–JMC

14.1 ± 1.0 11.9 ± 1.1 ( 16)

145.80 ± 3.0 89.38 ± 2.0* ( 38)

15.1 ± 1.1 13.1 ± 1.1 ( 13)

151.51 ± 5.0 104.33 ± 4.0* ( 31)

12.3 ± 1.1 9.6 ± 0.9 ( 22)

126.33 ± 3.0 53.33 ± 2.0* ( 58)

30.1 ± 2.0 24.3 ± 2.0 ( 19)

350.00 ± 11 190.83 ± 12* ( 45)

29.8 ± 2.0 25.2 ± 1.6 ( 15)

300.3 ± 18 186.3 ± 11* ( 38)

56.2 ± 3.0 44.4 ± 1.7 ( 21)

600.5 ± 22 125.16 ± 10* ( 79)

25.4 ± 2.0 20.1 ± 1.6 ( 20)

256.3 ± 12 161.3 ± 10* ( 37)

29.4 ± 2.1 25.6 ± 2.0 ( 13)

300.0 ± 18 204.0 ± 8* ( 32)

20.10 ± 3.0 15.80 ± 0.09 ( 21)

201.50 ± 22 100.60 ± 7* ( 50)

1.9 ± 0.09 1.5 ± 0.05 ( 20)

18.30 ± 1.2 10.81 ± 1.0* ( 40)

1.4 ± 0.08 1.1 ± 0.09 ( 21)

14.50 ± 1.0 10.20 ± 0.9* ( 30)

3.8 ± 0.22 3.0 ± 0.21 ( 20)

37.00 ± 3.0 14.65 ± 1.4* ( 65)

Results are mean ± SEM of five different experiments. Enzyme specific activities are expressed as lmoles/mg protein/h. Values in parenthesis represent percent change from respective controls. * Significantly different at p < 0.05 as compared to saline-treated control.

3.3. Effect of UN on the activities of certain enzymes of carbohydrate metabolism The main function of kidney i.e. reabsorption of various ions and solutes depends on the continuous energy supply as ATP which is generated by various metabolic pathways including glycolysis and oxidative metabolism. The acute renal failure produced by toxic insult results in reduced oxygen consumption due to damage caused to mitochondria and other organelles. To determine the effect of UN on the enzymes of various metabolic pathways, the kidneys were harvested from UN treated rats and the homogenates were prepared from cortex (WC) and medulla (M). The cortex is further divided into superficial (SC) and juxtamedullary cortex (JMC) to ascertain the specificity of the

effects. The activities of lactate dehydrogenase (LDH) (glycolysis); isocitrate ICDH, succinate (SDH) and malate (MDH) dehydrogenases, (TCA cycle); glucose-6-phosphatase (G6Pase), fructose-1,6-bisphosphatase (FBPase), (gluconeogenesis); and glucose-6-phosphate dehydrogenase (G6PDH) (HMP shunt pathway) and NADP-malic enzyme were determined in various homogenates. UN treatment caused a significant increase in LDH activity both in the cortex (+18%) and to a greater extent in medulla (+31%) compared with respective control values. Further analysis showed that increase in LDH activity was much greater in JMC (+36%) compared with SC (+20%) homogenates (Table 5). In contrast, the activities of ICDH, SDH and MDH significantly declined in response to UN treatment in WC, M, SC and JMC

2084

A.A. Banday et al. / Food and Chemical Toxicology 46 (2008) 2080–2088

Table 4 Effect of 5 days UN treatment on kinetic parameters of BBM-AlkPase, GGTase, maltase and LAP isolated from whole cortex (BBMV–WC), superficial cortex (BBMV–SC) and juxtamedullary cortex (BBMV–JMC) Group

WC

SC

Vmax

Km (10

AlkPase Control UN

150.00 ± 7 107.00 ± 6*

GGTase Control UN Maltase Control UN LAP Control UN

3

JMC

Vmax

Km (10

0.114 ± 0.005 0.117 ± 0.007

250.00 ± 10 166.66 ± 6*

250.00 ± 12 200.00 ± 8*

0.800 ± 0.040 0.780 ± 0.030

157.90 ± 8 112.80 ± 6*

20.00 ± 1.0 20.10 ± 1.8

18.75 ± 1.3 13.04 ± 0.6*

M)

0.400 ± 0.020 0.390 ± 0.020

3

3

Vmax

Km (10

0.190 ± 0.009 0.200 ± 0.010

156.25 ± 8 93.75 ± 5*

0.200 ± 0.009 0.200 ± 0.010

272.15 ± 15 214.30 ± 11*

0.570 ± 0.001 0.570 ± 0.001

500.00 ± 20 200.00 ± 8*

0.800 ± 0.010 0.800 ± 0.001

358.85 ± 15 272.72 ± 15*

10.00 ± 0.5 9.85 ± 0.5

187.50 ± 8 93.75 ± 4*

13.33 ± 0.5 13.28 ± 0.7

17.75 ± 1.1 12.93 ± 0.6*

M)

0.400 ± 0.018 0.410 ± 0.020

38.46 ± 2.0 20.00 ± 1.3*

M)

0.260 ± 0.020 0.260 ± 0.021

Values are mean ± SEM of five different experiments. Values are calculated from Lineweaver–Burk plots. * Significantly different at p < 0.05 as compared to saline-treated control.

homogenates (WCH, MH, SCH,JMCH) (Table 5). The effect was greater in medullary than in cortical enzymes. Further, the activities of ICDH, SDH and MDH declined to a greater extent in JMCH compared with SCH by UN treatment. The effect of UN was also determined on the activities of G6Pase and FBPase, representative enzymes of gluconeogenesis. The activities of G6Pase and FBPase reduced significantly similarly in both cortex and medulla (Table 5). However the decrease in activity was relatively more in SCH than JMCH. The effect of UN was differentially observed on the activities of G6PDH and malic enzyme, sources of cellular NADPH used for biosynthetic activity. In UN treated rats, the activity of G6PDH significantly decreased both in the cortex ( 36%) and medulla ( 42%) but to greater extent in MH. In contrast to G6PDH, the activity of malic enzyme (ME) significantly increased in the cortex (+30%) as well as in medulla (+40%). The activity of both the enzymes similarly altered in SCH and JMCH. The decrease in G6PDH and increase in ME was relatively greater in JMCH compared with SCH (Table 5). In addition to carbohydrate metabolism enzymes, the activities of Na+–K+ ATPase (basolateral membrane enzyme) and AcidPase (lysosomal enzyme) were also significantly affected both in cortex and medulla by UN administration. The activity of Na+–K+ ATPase significantly decreased whereas the activity of AcidPase significantly increased in UN treated compared to control rats (Table 6). The effect of UN on both the enzymes appeared to be greater in medulla than cortex and in JMCH compared to SCH. 3.4. Effect of UN on the antioxidant defense system It is evident that reactive oxygen species (ROS) generated by various toxins are important mediators of cell injury and pathogenesis of renal diseases especially in the

kidney. It is well established that kidney has an effective defense system necessary to control ROS metabolism. This system includes the antioxidant enzymes such as Cu, Znsuperoxide dismutase, catalase and glutathione peroxidase (GPx). The effect of UN was observed on lipid peroxidation, total-SH and on the activities of above enzymes (Table 7) in whole cortex to understand the role of oxidative stress in UN induced nephrotoxicity. Lipid peroxidation measured in terms of malondialdehyde (MDA) levels significantly increased (+51%) whereas total-SH significantly declined ( 29%) by UN treatment. The activities of Cu, Zn-SOD and glutathione oxidase significantly increased, however, activity of catalase decreased ( 35%). It appears that UN induced nephrotoxicity perturbed the antioxidant defense system. 4. Discussion Uranium is a heavy metal that invariably carries an exposure risk for industrial workers as well as general population. The extensive use of depleted uranium in both civilian and military applications has increased the number of human beings exposed to this compound. Histopathological examinations showed severe damage to kidney and liver by UN exposure (Haley et al., 1982). Recently renal toxicogenomic studies revealed that UN causes alteration of many genes involved in cellular metabolism, oxidative stress, signal transduction etc (Taulan et al., 2004). However, biochemical correlations of these effects remained to be unravelled. The present investigation was aimed to determine multifaceted effects of a single nephrotoxic dose of UN on the activities of various enzymes involved in glycolysis, TCA cycle, gluconeogenesis, HMP shunt pathway, antioxidant defense system and enzymes of renal proximal tubular BBM and BLM to understand mechanism of its nephrotoxic actions. The nephrotoxic effect of UN was

A.A. Banday et al. / Food and Chemical Toxicology 46 (2008) 2080–2088

C

*- 67%

* + 94%

UN

C

* + 178%

JMC

SC

UN

JMC

200

*- 70%

* +85.3%

300

* - 48%

400

* + 96%

500

100

* + 130%

600

* - 55%

0

C

C

UN

C

UN

UN

UN

+ 80%

- 50%

JMC

- 35%

C

* + 171%

*- 63%

C

SC

- 37%

45 40 35 30 25 20 15 10 5 0

UN

JMC

C

WC

D Total enzyme units

SC

*- 45%

200 180 160 140 120 100 80 60 40 20 0

C

* - 40%

WC

C

UN

UN

C

+120%

UN

* + 90%

C

* + 96%

Total enzyme units

UN

WC

B

Total enzyme units

* +109.09%

* -51.11%

C

+ 85%

Total enzyme units

180 160 140 120 100 80 60 40 20 0

SC

* - 45%

WC

A

UN

Fig. 1. Total enzyme activities of (A) AlkPase, (B) GGTase, (C) maltase and (D) LAP in homogenates of whole cortex (WC), superficial cortex (SC) and Juxtamedullary cortex (JMC) from UN and saline-treated control rats (h) – bound, (j) – free. Values are represented as mean ± SEM of five different preparations. *Significantly different from control at p < 0.01 or higher degree by one-way ANOVA.

manifested by increased serum creatinine, elevated BUN accompanied by proteinuria and decreased creatinine clearance associated with increased loss of various electrolytes (Na+, K+, Ca2+, Mg2+ and Pi) and excretion of various membrane-associated enzymes indicating that a significant kidney damage has occurred. UN caused increase in serum cholesterol and phospholipids. A positive balance of cholesterol and phospholipids (essential membrane components) in serum may facilitate repair and regulation of the membrane as required after UN exposure. The activities of BBM markers enzymes were determined to examine the structural/functional damage caused

2085

by UN exposure to BBM integrity. The activities of AlkPase, maltase, GGTase and LAP markedly declined in BBMV isolated from WC, SC and JMC but not changed significantly in respective homogenates in UN treated compared to control rats. Further analyses of homogenates revealed that enzyme activities were actually decreased in the pellet of homogenate (membrane bound fractions) whereas increased proportionally in the supernatants obtained after centrifugation (Fig. 1). This implies that BBM was severely damaged and may have been partially effaced by toxic UN insult as evident by histopathological studies (McDonald-Taylor et al., 1997; Gilmen et al., 1998). Infact the enzymes and proteinic components were dissociated from the membrane and excreted in the urine as observed during ischemic injury (Khundmiri et al., 1997). Our data is in partial agreement with those of previously reported human studies in which urinary glucose, alkaline phosphatase and b2 microglobulin were found to be increased upon chronic uranium intoxication (Kurttio et al., 2002). The observation that UN caused greater decrease of enzyme activities in BBMV–JMC compared to BBMV–SC is consistent with the fact that a greater damage occurred to pars recta (S3 sub segments of proximal tubule) located in juxtamedullary cortex in comparison to pars convoluta (S1 segments) located in superficial cortical region (Haley et al., 1982; Gilmen et al., 1998; Sun et al., 2000). UN induced greater decrease in activities of GGTase and LAP compared to AlkPase and maltase further supports that UN has caused greater damage to S3 sub segments of proximal tubule as GGTase and LAP are known biomarkers of S3 sub segments (Yusufi et al., 1994). Kinetic studies further strengthen this viewpoint as the decrease in the marker enzyme activities was largely due to decrease in Vmax, with little or no effect on Kmvalues indicating reduction/loss of active enzyme molecules from the membrane. Nephrotoxicants such as cisplatin, mercury and renal ischemia have been shown to damage S3 sub segments (Khundmiri et al., 1997; Fatima et al., 2004; Barbier et al., 2005) whereas gentamicin and K2Cr2O7 showed greater damage to S1 and S2 of proximal tubules (Cronin et al., 1986; Fatima et al., 2005). UN is presumed to cause renal damage because of interactions with enzyme protein sulphydryl group in the acidic luminal membrane and/or depletion of intracellular glutathione (Weiner and Jacobs, 1983) causing phospholipids derangement and alteration in membrane permeability. It has been reported in yeast cells that uranyl ion interacts with cell membranes in acidic environment forming stable complexes with phosphoryl and carbonyl ligands (Wilson et al., 1984). Since the pH of luminal fluid in proximal straight tubule is relatively more acidic than in proximal convoluted tubule; UN appears to interact and accumulate preferentially in pars recta. The reabsorption of Na+ by proximal tubular BBM is considered to be the major function of the kidney because the transport of other ions and various solutes depends directly or indirectly on Na+ reabsorption (Coux et al.,

2086

A.A. Banday et al. / Food and Chemical Toxicology 46 (2008) 2080–2088

Table 5 Effect of UN treatment on the activities of LDH, SDH, MDH, ICDH, G6Pase, FBPase, G6PDH and ME in the homogenates of whole cortex (WC), medulla (M), superficial cortex (SC) and juxtamedullary cortex (JMC) Group

WC

M

Control

UN *

LDH

22.0 ± 0.5

SDH

12.21 ± 0.5

MDH

112.4 ± 3.0

ICDH

8.5 ± 0.3

G6Pase

0.32 ± 0.01

FBPase

0.87 ± 0.07

G6PDH

1.3 ± 0.06

ME

13.08 ± 0.4

SC

Control

25.96 ± 0.4 (+18) 7.94 ± 0.2* ( 35) 99.8 ± 2.0* ( 11) 6.29 ± 0.25* ( 26) 0.23 ± .01* ( 27) 0.6 ± 0.03* ( 31) 0.83 ± 0.04* ( 36) 17.00 ± 0.6* (+30)

UN *

30.0 ± 0.6 9.1 ± 0.4 100.0 ± 2.0 6.2 ± 0.2 0.19 ± 0.01 0.75 ± 0.07 0.95 ± 0.04 9.2 ± 0.3

Control

39.3 ± 1.0 (+31) 5.46 ± 0.3* ( 40) 74.0 ± 1.5* ( 26) 3.8 ± 0.12* ( 38) 0.13 ± 0.01* ( 32) 0.4 ± 0.02* ( 46) 0.55 ± 0.02* ( 42) 12.9 ± 0.4* (+40)

21.4 ± 0.4 14.0 ± 0.90 107.0 ± 5.0 9.5 ± 0.12 0.35 ± 0.02 0.86 ± 0.08 1.32 ± 0.04 13.4 ± 0.33

JMC UN *

25.68 ± 0.6 (+20) 9.8 ± 0.68* ( 30) 94.2 ± 3.0* ( 12) 7.1 ± 0.3* ( 25) 0.23 ± 0.01* ( 35) 0.58 ± 0.05* ( 32) 0.86 ± 0.04* ( 35) 17.70 ± 0.6* (+31)

Control

UN

29.85 ± 0.6

40.4 ± 1.0* (+36) 4.6 ± 0.2* ( 45) 73.6 ± 2.0* ( 27) 3.60 ± 0.2* ( 40) 0.15 ± 0.001* ( 42) 0.48 ± 0.04* ( 36) 0.5 ± 0.02* ( 46) 13.06 ± 0.24* (+40)

8.5 ± 0.8 100.3 ± 3.0 6.00 ± 0.15 0.26 ± 0.01 0.75 ± 0.05 0.93 ± 0.03 9.20 ± 0.31

Results are mean ± SEM of five different experiments. Enzyme specific activities are expressed as lmoles/mg protein/h. Values in parenthesis represent percent change from respective controls. * Significantly different at p < 0.05 as compared to saline-treated control.

Table 6 Effect of UN treatment on the activities of Na+–K+ ATPase and AcidPase in the homogenates of whole cortex (WC), medulla (M), superficial cortex (SC) and juxtamedullary cortex (JMC) Group WC Control UN M Control UN SC Control UN JMC Control UN

Na+–K+ ATPase

AcidPase

5.06 ± 0.3 3.84 ± 0.2* ( 24%)

10.27 ± 0.4 12.22 ± 0.46* (+19%)

4.40 ± 0.2 2.86 ± 0.1* ( 35%)

6.15 ± 0.3 7.62 ± 0.28* (+24%)

5.30 ± 0.25 3.71 ± 0.3* ( 30%)

10.5 ± 0.04 12.7 ± 0.45* (+21%)

4.89 ± 0.4 2.90 ± 0.12* ( 40%)

7.20 ± 0.29 8.90 ± 0.22* (+24%)

Results are mean ± SEM of five different experiments. Enzyme specific activities are expressed as lmoles/mg protein/h. Values in parenthesis represent percent change from respective controls. * Significantly different at p < 0.01 as compared to saline-treated control.

2001). Since these transports depend on structural integrity of BBM and available energy as ATP which is supplied by various metabolic pathways it is imperative that any alterations to these pathways caused by toxic insults would determine the rate of renal transport functions (Khundmiri et al., 2004, 2005). As shown in Section 3, the activities of various enzymes involved in glycolysis, TCA cycle, gluconeogenesis and HMP shunt pathway were affected by UN

treatment albeit differently. UN significantly increased the activity of LDH in all renal tissues studied with greater increase observed in the medulla and JMCH compared to WCH and SCH. However, the activities of ICDH, SDH and MDH, enzymes of TCA cycle significantly decreased, more in MH and JMCH than in WCH and SCH by UN administration. The marked decrease in ICDH, SDH and MDH activities indicate an impaired oxidative metabolism of glucose and decreased ATP production and hence depressed renal transport function. Although the actual rates of glycolysis and other pathways were not determined, the increased activity of LDH along with simultaneous decline in ICDH, SDH and MDH may suggest a shift in energy production from mitochondria to glycolysis due to mitochondrial damage caused by UN (McDonaldTaylor et al., 1997; Gilmen et al., 1998). The decrease in activities of gluconeogenic enzymes: FBPase and G6Pase may be the result of decrease in TCA cycle enzyme activities. This can be further explained by the fact that the reduced activities of TCA cycle enzymes especially that of MDH may have reduced the production of oxaloacetate from malate which is required not only for the continuation of TCA cycle but also for gluconeogenesis. The oxidative conversion of glucose or glucose-6-phosphate to 6-phosphogluconate by HMP shunt pathway, which is catalyzed by G6PDH, was also found to be lowered due to mitochondrial dysfunction by UN treatment. The lower generation of NADPH by G6PDH, which is essential for many reductive anabolic reactions, may have been compensated to some extent by increased activity of NADP-malic enzyme. The decrease in Na+–K+ ATPase activity, showed that UN caused damage to basolateral membrane of proximal tubule. UN induced increase in acid

A.A. Banday et al. / Food and Chemical Toxicology 46 (2008) 2080–2088

2087

Table 7 Effect of UN treatment on lipid peroxidation, total-SH, SOD, catalase and glutathione peroxidase Group

Lipid peroxidation

Total SH

SOD

Catalase

Glutathione peroxidase

Control UN

181.94 ± 4.82 273.950 ± 3.46* (+51%)

4.14 ± 0.29 2.946 ± 0.06* ( 29%)

14.39 ± 0.46 22.020 ± 0.05* (+53%)

217.00 ± 2.62 141.36 ± 2.35* ( 35%)

0.2115 ± 0.01 0.278 ± 0.01* (+32%)

Results are mean ± SEM of five different experiments. Values in parenthesis represent percent change from respective controls. Lipid peroxidation is measured in nanomoles/g tissue, total-SH in lmoles/g tissue. SOD is measured in units/mg protein (unit – the amount which causes 50% inhibition of pyrogallol oxidation in a reaction volume of 3 ml.) Catalase and glutathione peroxidase in lmoles/mg/min. * Significantly different at p < 0.01 as compared to saline-treated control. Damage to proximal tubular membrane Decrease in G6PDH activity Decreased NADPH production

Mitochondrial dysfunction

Effacement and interiorization of BBMV Decrease in AlkPase; GGTase; LAP; Maltase activities Decrease in Na+-K+ATPase activity

Damage caused

Oxidative stress

Loss of TCA cycle enzymes (ICDH, MDH, SDH)

Increased lipid peroxidation Increased SOD and GPx activity

URANYL NITRATE Kidney Response

phosphatase activity indicate an increased number and size of lysosomes in proximal tubule epithelial cells as reported by histopathological studies by UN exposure (Haley et al., 1982). Reactive oxygen species and other free radicals are considered important mediators of renal cell injury by toxic insult and renal ischemia (Walker et al., 1999; Taulan et al., 2004). Glutathione (GSH) and its redox cycle enzymes represent an important cellular defense system against oxidative stress (Kaplowitz et al., 1985). A significant increase in LPO and alterations in activities of SOD, catalase and GPx are indicative of UN induced oxidative balance perturbation (Schramm et al., 2002). It appears that while increased SOD activity causes dismutation of generated superoxide radicals whereas increased glutathione peroxidase scavenges H2O2 and lipid peroxides produced as a result of UN exposure. Our present biochemical observations indicate a positive correlation with recently published toxicogenomic studies (Taulan et al., 2004). These toxicogenomic studies revealed up-regulation of several genes or their products including those of cellular metabolism, oxidative stress and cellular membranes by UN exposure. Of particular interest was the up-regulation of genes of enzymes involved in glycolysis (LDH, aldolase, phosphoglycerokinase), in NADPH production (NADP-malic enzyme, NADP–ICDH) and in oxidative stress (SOD, GPx) and down-regulation of Na+–K+ ATPase. Taken together, the observations provide useful information to identify new putative biomarkers of UN induced nephrotoxicity. In contrast to UN, gentamicin and cisplatin have been shown to cause downregulation of many of these genes. It appears that UN, GM and cisplatin may exert nephrotoxic effects leading to ARF by different mechanisms. In conclusion, the results of the present study indicate that UN elicited deleterious nephrotoxic effects (as summarized in Fig. 2) by causing major damage to proximal tubular plasma membranes (both BBM and BLM), and mitochondria as reflected by significant decrease in the activities of specific biomarker enzymes of these organelles. UN caused greater effects to juxtamedullary compared to superficial cortex indicating predominant damage to S3 sub segments of proximal tubules located in the JMregion. UN seems to enhance glycolytic enzyme LDH in order to increase energy dependence on glycolysis due to

Shift to Glycolysis for energy production Increased LDH activity

Increase in NADPH production by NADP- Malic enzyme NADP- ICDH

Gene Expression Up regulation of NADP-ICDH, NADP-ME, Aldolase, PC-Kinases Down regulation of Na+-K+ATPase

Fig. 2. Uranyl nitrate induced acute renal failure: mechanism.

depressed TCA cycle enzymes. UN caused some of these effects; atleast in part, by ROS mediated mechanism. The parallel changes observed in our studies with those of toxicogenomic studies may provide a basis for determination of novel potential biomarkers in addition to traditional measures (creatinine, BUN) of renal injury due to heavy metals including UN. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements Research grant (SP/SO/B-93/89) from Department of Science and Technology (DST), Government of India to ANKY. and to the department from University Grants Commission (UGC-DRF, DST – FIST) is gratefully acknowledged. AAB and NF are recipients of fellowship from Council of Scientific and Industrial Research and SP from Indian Council of Medical Research, New Delhi, India.

2088

A.A. Banday et al. / Food and Chemical Toxicology 46 (2008) 2080–2088

References Anthony, M.L., Gartland, K.P.R., Beddell, C.R., Lindon, J.C., Nicholson, J.K., 1994. Studies of the biochemical toxicology of uranyl nitrate in the rat. Arch. Toxicol. 68, 43–53. Barbier, O., Jacquillet, G., Tauc, M., Cougnan, M., Poujeol, P., 2005. Effect of heavy metals on, and handling by kidney. Nephron Physiol. 99, 105–110. Bosshard, E., Zimmerli, B., Schalatter, C., 1992. Uranium in diet: risk assessment of its nephro- and radiotoxicity. Chemosphere 24, 309– 321. Coux, G., Trurumper, L., Elias, M.M., 2001. Cortical Na+–K+ ATPase activity, abundance and distribution after in vivo renal ischemia without reperfusion in rats. Nephron 89, 82–89. Cronin, R.E., Brown, D.M., Simonsen, R., 1986. Protection by thyroxine in nephrotoxic acute renal failure. Am. J. Physiol. 25, F408–F416. Cronin, R.E., Thompson, J.R., 1991. Role of potassium in the pathogenesis of acute renal failure. Miner. Electrol. Metab. 17, 100–105. Domingo, J.L., 1994. Metal induced developmental toxicity in mammals: a review. J. Toxicol. Environ. Health 42, 123–141. Farooq, N., Priyamvada, S., Arivarasu, N.A., Salim, S., Khan, F., Yusufi, A.N.K., 2006. Influence of Ramadan type fasting on enzymes of carbohydrate metabolism and brush border membrane in small intestine and liver of rat used as a model. Brit. J. Nutr. 96, 1087– 1094. Fatima, S., Yusufi, A.N.K., Mahmood, R., 2004. Effect of cisplatin on renal brush border membrane enzymes and phosphate transport. Hum. Exp. Toxicol. 23, 547–554. Fatima, S., Arivarasu, N.A., Banday, A.A., Yusufi, A.N.K., Mahmood, R., 2005. Effect of potassium dichromate on renal brush border membrane enzymes and phosphate transport in rats. Hum. Exp. Toxicol. 24, 631–638. Flohe, L., Gunzler, W., 1984. Assay of glutathione peroxidase. In: Colowick, S.P., Kaplan, N.O. (Eds.), Methods Enzymology, vol. 105. Academic Press, New York, pp. 114–121. Giri, U., Iqbal, M., Athar, M., 1996. Porphyrin-mediated photosensitization has a weak tumor promoting activity in mouse skin: possible role of in situ generated reactive oxygen species. Carcinogenesis 17, 2023–2028. Gilmen, A.P., Villeneuve, D.C., Secours, V.E., Yagminas, A.P., Tracyl, B.L., Quinn, J.M., Valli, V.E., Yagminas, A.P., Tracy, B.L., Quinn, J.M., Valli, V.E., Wiles, R.J., Moss, M.A., 1998. Uranyl nitrate: 28day and 91-day toxicity studies in the Sprague–Dawley rat. Toxicol. Sci. 41, 117–128. Goldman, M., Yaari, A., Doshnitzki, Z., Cohen-Luria, R., Moran, A., 2006. Nephrotoxicity of uranyl acetate: effect on rat kidney brush border membrane vesicles. Arch. Toxicol. 80, 387–393. Haley, D.P., Bulger, R.E., Dobyan, D.C., 1982. The long term effects of uranyl nitrate on the structure and function of the rat kidney. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 41, 181–192. Kaplowitz, N.T., Aw, Y., Ookhtens, M., 1985. The regulation of hepatic glutathione. Annu. Rev. Pharmacol. Toxicol. 25, 715–774. Khundmiri, S.J., Asghar, M., Khan, F., Salim, S., Yusufi, A.N.K., 1997. Effect of reversible and irreversible ischemia on marker enzymes of BBM from renal cortical PT subpopulations. Am. J. Physiol. 273, F849–F856. Khundmiri, S.J., Asghar, M., Banday, A.A., Khan, F., Salim, S., Levi, M., Yusufi, A.N.K., 2004. Effect of ischemia and reperfusion on enzymes of carbohydrate metabolism in rat kidney. J. Nephrol. 17, 377–383.

Khundmiri, S.J., Asghar, M., Banday, A.A., Khan, F., Salim, S., Levi, M., Yusufi, A.N.K., 2005. Effect of reperfusion on sodium dependent phosphate transport in renal brush border membranes. Biochim. Biophys. Acta 1716, 19–28. Kurttio, P., Auvinem, A., Salonem, L., Saha, H., Pekkanem, J., Makelainem, I., Vaisanen, S.B., Pentilla, I.M., Komulainen, H., 2002. Renal effects of uranium in drinking water. Environ. Health Perspect. 110, 337–342. McDonald-Taylor, C.K., Singh, A., Gilman, A., 1997. Uranyl nitrateinduced proximal tubule alterations in rabbits: a quantitative analysis. Toxicol. pathol. 25, 381–389. Marklund, S., Marklund, G., 1974. Involvement of the superoxide anion radical in the auto oxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 47, 469–474. Miller, A.C., Fueiarelli, A.F., Jackson, W.E., Ejnik, E.J., Emond, C., Strocko, S., Hogan, J., Page, N., Pellmar, T., 1998. Urinary and serum mutagenicity studies with rats implanted with depleted uranium or tantalum pellets. Mutagenesis 13, 643–648. Miller, A.C., Stewart, M., Brooks, K., Shi, L., Page, N., 2002. Depleted uranium-catalyzed oxidative DNA damage: absence of significant alpha particle decay. J. Inorg. Biochem. 91, 246–252. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351– 358. Sanchez, D.Z., Belles, M., Albina, M.L., Sirvent, J.J., Domingo, J.L., 2001. Nephrotoxicity of simultaneous exposure to mercury and uranium in comparison to individual effects of these metals in rats. Biol. Trace Elem. Res. 84, 139–154. Schramm, L., La, M., Heidbreder, E., Hecker, M., Beckman, J.S., Lopau, K., 2002. L-Arginine deficiency and supplementation in experimental acute renal failure and in human kidney transplantation. Kidney Int. 61, 1423–1432. Sedlak, J., Lindsay, R.H., 1968. Estimation of total protein bound and non protein bound SH groups in tissue with Ellman’s reagent. Anal. Biochem. 25, 192–205. Stochs, S.J., Bagchi, D., Hassoun, E., Bagchi, M., 2000. Oxidative mechanisms in the toxicity of chromium and cadmium ions. J. Environ. Pathol. Toxicol. Oncol. 19, 201–213. Sun, D.F., Fujigaki, Y., Fujimoto, T., Yonemura, K., Hishida, A., 2000. Possible involvement of myfibroblasts in cellular recovery of uranyl acetate-induced acute renal failure in rats. Am. J. Pathol. 157, 1321– 1324. Taulan, M., Paquet, F., Maubert, C., Delissen, O., Demaille, J., Romey, M.C., 2004. Renal toxicogenomic response to chronic uranyl nitrate insult in mice. Environ. Health Perspect. 112, 1628–1635. Walker, P.D., Barri, Y., Shah, S.V., 1999. Oxidant mechanisms on gentamicin nephrotoxicity. Renal Failure 21, 433–442. Weiner, M.W., Jacobs, C., 1983. Mechanism of cisplatin nephrotoxicity. Fed. Proc. 42, 2974–2978. Wilson, D.R., Arnold, P.E., Burke, T.G., Schrier, R.W., 1984. Mitochondrial calcium accumulation and respiration in ischemic acute renal failure in the rat. Kidney Int. 25, 519–526. Yusufi, A.N.K., Murayama, N., Gapstur, S.M., Szczepanska-Konkel, M., Dousa, T.P., 1994. Differential properties of brush border membrane vesicles from early and late proximal tubules of rat kidney. Biochim. Biophys. Acta. 1191, 117–132. Zhong, L.F., Zhang, J.G., Zhang, M., Ma, S.L., Xia, Y.X., 1990. Protection against cisplatin-induced lipid peroxidation and kidney damage by procaine in rats. Arch. Toxicol. 64, 599–600.