The Renal Effects of Vanadate Exposure: Potential Biomarkers and Oxidative Stress as a Mechanism of Functional Renal Disorders—Preliminary Studies

The alterations in the levels/activities of selected biomarkers for detecting kidney toxicity and in the levels of some oxidative stress (OS) markers and elements were studied in male rats to evaluate biochemically the degree of kidney damage, investigate the role of OS in the mechanism of functional renal disorders, reveal potential biomarkers of renal function, and assess the renal mineral changes in the conditions of a 12-week sodium metavanadate (SMV, 0.125 mg V/mL) exposure. The results showed that OS is involved in the mechanism underlying the development of SMV-induced functional renal disturbances. They also suggest that the urinary cystatin C (CysCu) and kidney injury molecule-1 (KIM-1u) could be the most appropriate to evaluate renal function at the conditions of SMV intoxication when the fluid intake, excreted urinary volume (EUV), body weight (BW), and the urinary creatinine excretion (Creu) decreased. The use of such tests as the urinary lactate dehydrogenase, alkaline phosphatase, γ-glutamyltranspeptidase, and N-acetyl-β-D-glucosaminidase (LDHu, ALPu, GGTPu, and NAGu) seems not to be valid given their reduced activities. The use of only traditional biomarkers of renal function in these conditions may, in turn, be insufficient because their alterations are greatly influenced by the changes in the fluid intake and/or BW.


Introduction
Vanadium (V) is a well-known powerful prooxidant. It may modify oxidative stress (OS) in the cells and be involved in oxidative injury mechanisms [1]. Its prooxidant action has been demonstrated in in vivo and in vitro conditions [2][3][4][5][6][7]. It has been shown that the free radical processlipid peroxidation (LPO), which is a biochemical biomarker of cellular dysfunction and an index of cytotoxicity [8], is enhanced by V in the kidney [3,8]. It has also been suggested that LPO may be predictive of renal dysfunction [8].
Kidney is particularly vulnerable to deleterious effects of V. The susceptibility of this organ to V may be a reflection of its accumulation in this tissue [8]. It has been reported that V may be present in tubular cells in a readily exchangeable form as well as in low and high molecular mass complexes, and it may be excreted free or bound to proteins after prolonged exposure [9,10]. It has also been speculated that V may be involved in the pathogenesis of distal renal tubular acidosis (dRTA), renal stone disease, "uremic syndrome, " and acquired cystic kidney disease [11][12][13]. A suggestion that prolonged intake of high-dose V supplements may cause serious kidney toxicity has been put forward as well [14,15].

BioMed Research International
The occupational and environmental toxicological impact of V and the fact that kidneys are critical for its poisoning [16][17][18][19], the modest number of biomarkers of possible functional renal disturbances under vanadate exposure examined until now, and the insufficient information about the contribution of OS in the mechanism underlying the vanadate-induced functional kidney disorders prompted us to answer the following questions. (a) To what extent do the effects of 12-week sodium metavanadate (NaVO 3 , SMV, 0.125 mg V/mL) exposure alter the levels/activities of some biomarkers of renal toxicity in rats? We intended to examine traditional biomarkers classified as not very specific or sensitive but routinely used in the diagnosis of kidney function and those that allow distinguishing, to some degree, structural and functional renal disorders and have a potential for determining the site of renal tubular damage [20][21][22]. (b) To what degree do the effects of SMV exposure change the homeostasis of some micro-and macroelements in the kidney? (c) Which of the biomarkers examined are the most sensitive in our experimental conditions? (d) What value/information do some biomarkers add to the existing data? (e) Is OS involved in the mechanism of the development of kidney function disorders during SMV intoxication? (f) Are there any significant relationships between the measured variables? Since the measurement of the activities of some enzymes in tissues and biological fluids may play a significant role in detection of tissue cellular injury and point to damage long before histological alterations, both cytosolic and lysosomal enzymes as well as those located on the brush-border membrane have been taken into consideration and illustrated in the present report.

Reagents.
The kits for determination of the plasma concentrations of TP p , U p , UA p , Mg p , and Ca p and the urinary levels of U u , UA u , and Cre u were obtained from Emapol (Gdańsk, Poland), whereas the kits for determination of the plasma Cu p and Zn p levels were purchased from Sentinel Ch. kits (Milan, Italy). The reagents for determination of the levels/activities of the plasma (Cre p , ALB p , LDH p , GGTP p , and ALP p ), urinary (ALB u , TP u , LDH u , GGTP u , and ALP u ), and renal (LDH k , GGTP k , and ALP k ) biomarkers as well as the reagent for measurement of the levels of electrolytes in the plasma and urine (Na p/u , K p/u , and Cl p/u ) were acquired from Alpha Diagnostics (Warsaw, Poland). The kit for estimation of total antioxidant status in the kidney (TAS k ) was bought from Calbiochem-Novabiochem Corporation (San Diego, CA, USA), whereas the kits for determination of the urinary levels of NAG u (E90069Ra), CysC u (E90896Ra), 2 M u (E90260Ra), and KIM-1 u (E90785Ra) and of the plasma concentrations of CysC p (E90896Ra) and ( 2 M p E90260Ra) were derived from Uscn Life Science Inc. (Wuhan, China). NaVO 3 , Triton X-100, and the caesium chloride lanthanum chloride buffer (CsClLaCl) were purchased from Sigma Chemical (St. Louis, USA). Nitric acid (HNO 3 , 65% suprapure) and diethyl ether (C 4 H 10 O) were acquired from Merck (Darmstadt, Germany), whereas hydrogen peroxide (H 2 O 2 ; 30% pure P.A.) and the physiological buffered saline (PBS) were purchased from POCH (Gliwice, Poland) and from the Serum and Vaccine Factory (Biomed, Lublin, Poland), respectively. Stocks of V, Mg, Zn, and Cu (Inorganic Ventures, Christiansburg, USA) and a stock of Ca (Spectracer, UK) atomic absorption standard solutions as well as a multielement standard solution for Na and K (Sigma-Aldrich) were used in the element analysis by the Atomic Absorption Spectrometry (AAS) method. Ultrapure water was received from an ultrapure water HLP Spring 5R system 1 (Hydrolab, Gdańsk, Poland). All the chemicals were of the highest quality available.

Animals and Experimental
Design. The experiment was conducted according to the experimental protocol approved by the 1st Local Ethical Committee for Animal Studies in Lublin. Biological material used in this study originated from some of the outbred albino male Wistar rats used in the previous study [23]. The animals were kept in an animal room with controlled conventional conditions (one rat per stainless steel cage) and received, inter alia, deionised water (Group I, Control, 8 rats) or a water solution of NaVO 3 at a concentration of 0.125 mg V/mL (Group II, SMV, 8 rats) to drink in special bottles with the scale every day over a 12-week period. All the rats had ad libitum access to fresh deionised water, SMV solution, and a standard rodent diet (Labofeed B, Fodder and Concentrate Factory, Kcynia, Poland) in which the concentration of V had been assessed by Graphite Furnace AAS (GF-AAS) in our laboratory, and it was about 0.17 g V/g. More details about the chow had been provided by us previously [23]. The intake of food, water, and SMV solution was monitored daily and body weight (BW) was obtained weekly. The water and SMV solution intake was expressed as mL/rat/24 h, whereas the food intake was expressed as g/rat/24 h. The daily V intake in the SMVexposed rats was estimated based on the 24 h consumption of the drinking SMV solution and expressed as mg V/kg b. wt./24 h. During the whole experiment, the animals were observed in order to assess their general health. The doses of V presented in the study are within the broad dose range that was used for demonstrating the antidiabetic activity of V [19,24] and for analysing its pharmacokinetic behaviour [25] on an animal model. The concentration of V in rats' urine determined in the present study may reflect exposure to this element occurring especially in persons occupationally exposed to this metal [26,27].
Every second week in the course of the experiment and in the 11th week, 24-hour urine was collected from each rat placed individually in plastic metabolic cages not equipped with a cooling system (Tecniplast, Italy), which allowed separate collection of urine and faeces. During that time, each rat had access to food and water or the SMV solution. The urine samples from all the control and SMV-exposed rats were used immediately for determination of some biochemical parameters and for measurements of excreted urine volume (EUV) and urine pH. The volumes of excreted 24-hour urine were measured with a measuring cylinder, whereas urine pH and blood in the urine were tested on the H-100 urine analyser (DIRUI, China) using urinary dipsticks. Portions of the urine samples that were not used immediately were frozen at −80 ∘ C in a deep-freezer HFU 486 Basic 1 (Thermo Fisher Scientific, Germany) and stored until further analyses.
All the rats were sectioned at the end of week 12. Whole blood was taken from the jugular vein into plastic tubes with heparin as an anticoagulant under anaesthesia with ketamine/xylazine cocktail (100 mg/mL and 20 mg/mL, resp., i.p.) and centrifuged (5 min, 1500 ×g, 4 ∘ C). Plasma portions were collected for routine analyses of clinical chemistry parameters and for other biological determinations. The kidneys and femurs (right and left) were immediately removed. The kidneys were washed in ice-cold physiological saline (0.9% NaCl) and weighted. The right femurs (after removal of the overlying tissue with stainless steel knives) were also washed in 0.9% NaCl, weighed, and stored frozen at -80 ∘ C until the time of bone digestion. Before digestion, all the collected right femurs were cut using a diamond-disk saw (Metkon Micracut 175) 1 , cooled with ultrapure water to separate the proximal and distal femoral epiphysis (PFE and DFE) (a region of trabecular bone) from the femoral diaphysis (FD) (a region of cortical bone). Next, bone marrow was removed from all the FDs, which then were first soaked with ether (to remove fat content) and later in H 2 O 2 (to remove remaining blood deposits). Afterwards, the cleaned FDs were washed in ice-cold PBS and then in ultrapure water. Next, they were dried at room temperature into constant mass. The cleaned and bone marrow-deprived FD samples (∼0.355 g, mean) were used for digestion.

Decomposition of the Kidney, Urine, and FD.
In order to determine Mg, Ca, V, Zn, Cu, Na, and K in the kidney and Mg, Ca, V, Zn, and Cu in the urine, 0.5 g of kidney and 1 mL of urine were wet-mineralized with 5 mL of 65% HNO 3 in 12 Teflon Fluor Modified (TFM) closed digestion vessels using a model Speedwave Four microwave digestion system 1 (Berghof, Germany) equipped with a temperature and pressure sensor in each vessel. In turn, ∼0.355 g of FD was wet-mineralized in the same digestion system but in the presence of 5 mL of 65% HNO 3 and 1 mL of 30% H 2 O 2 to determine V, Mg, and Ca. Before the measurements, all the decomposed samples of kidney, FD, and urine were transferred into 25 mL volumetric polypropylene flasks by washing the inner surface of the digestion vessels with ultrapure water three times and filled up to the mark with ultrapure water. Next, determinations of the selected elements were performed by the AAS method.

Determination of Some Renal Biomarkers.
The concentrations of U p , UA p and TP p in the plasma as well as U u , UA u , and Cre u in 24-hour urine were determined colourimetrically by measuring the absorbance with a Thermo Spectronic BioMate5 UV-VIS spectrophotometer (UK). The concentrations of ALB p , ALB u , TP u , and Cre p and the activities of LDH p , GGTP p , ALP p , LDH u , GGTP u , and ALP u in the plasma and urine as well as the activities of LDH k , GGTP k , and ALP k in the kidney were measured using an automatic biochemical analyser BS-120 1 (Mindray, China). The levels of CysC p and 2 M p in the plasma and the levels of CysC u , 2 M u , KIM-1 u , and NAG u in the urine were assessed by a traditional method using rat-specific commercial enzymelinked immunosorbent assay (ELISA) kits and an ELISA microplate reader Synergy 2 1 equipped with an automated microplate strip washer ELx50 1 and a microplate shaker 1 (BIO-TEK Instruments Inc., USA). All the ELISA tests were performed according to the manufacturer's protocols. Before the measurements, thawed plasma and urine samples were mixed by inversion, centrifuged with cooling (1500 rpm for 10 min, 4 ∘ C, or 2000 rpm for 5 min, 4 ∘ C, resp.) using a centrifuge Heraeus Megafuge 11R 1 (Thermo Fisher Scientific, Germany), and immediately used for analysis. The optimal factors of dilution for some samples were chosen when necessary and the samples were diluted with ultrapure water or with PBS (pH 7.4 ± 0.2). In order to evaluate the glomerular filtration rate (GFR), calculation of creatinine clearance (CreC) was performed. The results for all the abovementioned parameters are expressed as % of the control and presented in Figures 1(a), 2(a), 2(b), 3(a), 3(b), and 4(a).

Spectrophotometric Determination of Ca, Mg, Zn, and
Cu in the Plasma. The measurements of these elements were performed using a BioMate5 spectrophotometer with the direct colourimetric method according to the protocols of the kits, and their concentrations are presented in Figures 1(b) and 1(d).

Determination of Na, K, and Cl in the Plasma and
Urine. The electrolytes mentioned were measured using an automatic EasyLyte analyser Na/K/Cl 1 (Medica). Their urinary excretion was first normalized against 24-hour diuresis and against 24-hour urinary Cre u excretion, and their concentrations are presented in Figures 1(b), 1(c), 2(e), and 3(e).

Atomic Absorption Measurements of Mg, Ca, V, Zn, Cu, Na, and K in Digested Kidney Samples, Mg, Ca, V, Zn, and Cu in Digested Urine Samples, V, Mg, and Ca in Digested FD
Samples, and V in Nondigested Plasma Samples. The elements were determined by Flame or Graphite Furnace AAS (F-AAS or GF-AAS, resp.) using a SpectrAA Z-2000 TANDEM atomic absorption spectrometer 1 (Hitachi, Japan) equipped with a Zeeman background corrector. A specific matrix modifier was used for V determination. In order to determine the V concentration in the plasma, the samples were diluted with 0.05% Triton X-100 and in ultrapure water when necessary. All the operating parameters of the instrument and the details of measurements of all the above-mentioned elements together with the values of the detection, quantification limits (LOD and LOQ, resp.), and the coefficient of variations (CV) are shown in Table 1.
The method of standard addition was performed in order to estimate the effect of interferences during Mg assessment. Mg, Ca, V, Zn, Cu, Na, and K were determined by application of a calibration curve using working standard solutions, which were obtained from a stock atomic absorption standard solutions containing 1000 g Mg, Ca, V, Zn, Cu, Na, and Table 1 Table 1. The plasma V level is presented in Figure 1(e). In turn, the urinary excretion of the above-mentioned elements investigated was also first normalized against 24-hour diuresis and against 24-hour urinary Cre u excretion. This is illustrated in Figures 2(c

Determination of Renal Lipid Peroxidation (LPO) and
Total Antioxidant Status (TAS). All details concerning the methodology of determination of LPO and the preparation of kidney homogenates and supernatants for the renal MDA k and TAS k measurements had already been provided [2,3]. The results of both the OS markers mentioned are expressed as % of control and illustrated in Figure 4(a).  test was applied when the data did not meet the assumptions of ANOVA. The results were presented as mean ± the standard error of the mean (SEM). A value less than 0.05 was taken as a criterion for a statistically significant difference. Pearson's correlation analyses were applied to assess the relationships between the measured variables. Correlations were considered statistically significant at < 0.05.

General Observation and Changes in Some Basic Parameters.
In the animals exposed to SMV, no distinct differences in physical appearance and motor behaviour were observed during the 12-week experimental period, compared with the control. Some of the SMV-intoxicated rats had gastrointestinal disturbances probably caused by the consumption of the V dose. One-day diarrhoea was observed in four animals from this group in the first, second, third, or fourth week of the experiment. Only one rat from this group had three-day diarrhoea in the first week of the experiment. The fluid and food intake as well as BW (Table 2) and EUV (Figure 2(a)) in the SMV-exposed rats were lower, compared with the control animals. The dose of V consumed by the rats during the 12week period reached the value of about 13 mg V/kg b. wt./24 h ( Table 2). (Blood and pH). The concentrations of Cre p and U p in the SMV-exposed rats increased (Figure 1(a)), whereas CreC (Figure 1(a)) and the levels of urinary excretion of Cre u (per 24 h) and U u (per 24 h) decreased significantly (Figure 2(a)), in comparison with the control animals. In turn, the urinary level of U u (per Cre u /24 h) (Figure 3(a)) and the concentration of U k in the kidney (Figure 4(a)) were unaltered. The levels of UA p (Figure 1(a)) and UA u (per 24 h and per Cre u /24 h, Figures  2(a) and 3(a), resp.) also remained unchanged in response to the SMV exposure.

Plasma, Urinary, and Renal Levels of the Examined Biomarkers and the Dipstick Urinalysis
The levels of TP p and ALB p in the SMV-exposed rats did not alter significantly, compared with the control (Figure 1(a)), but the urinary excretion of TP u (per 24 h)  and ALB u (per 24 h) decreased and unchanged, respectively, (Figure 2(a)). On the contrary, the urinary levels of TP u (per Cre u /24 h) in the same group of rats were unaltered and ALB u (per Cre u /24 h) increased, in comparison with the control (Figure 3(a)). In turn, the renal concentration of TP k and ALB k did not change markedly (Figure 4(a)). The concentrations of CysC p and 2 M p remained unaltered between the groups (Figure 1(a)). However, the urinary excretion of CysC u (per 24 h, Figure 2(a), and per Cre u /24 h, Figure 3(a)) increased markedly but the urinary 2 M u level (per 24 h, Figure 2(a), and per Cre u /24 h, Figure 3(a)) was unchanged. In turn, the urinary level of KIM-1 u (per 24 h, Figure 2(a), and per Cre u /24 h, Figure 3(a)) in the SMVintoxicated rats did not alter and increased, respectively, compared with the control.
The activity of LDH p increased, ALP p decreased, and GGTP p was unchanged in the rats after the SMV intoxication, in comparison with the control (Figure 1(a)). In turn, the  urinary activities of LDH u , GGTP u , and ALP u (per 24 h) as well as the urinary activities of GGTP u and ALP u (per Cre u /24 h) and NAG u (per 24 h and per Cre u /24 h) were lowered in the SMV-exposed rats, compared with the control (Figures 2(b) and 3(b)). However, the urinary activity of LDH u (per Cre u /24 h) only showed a visible trend toward an increase (Figure 3(b)). In turn, the renal activity of LDH k , GGTP k , and ALP k in the SMV-intoxicated rats was lowered, in comparison with the control (Figure 4(a)). Urine dipstick analysis did not reveal presence of blood in the urine of the SMV-intoxicated rats (data not shown). However, the urine pH in the same animals was markedly higher, compared with that in the control rats (Figure 2(a)).

Element Levels in the Plasma.
The exposure to SMV led to a distinct elevation of the Ca p and K p concentrations (Figure 1(b)), produced a visible decrease in the plasma Cu p level (Figure 1(d)), and increased the plasma V p concentration (Figure 1(e)). However, no SMV intoxication-related effects on the concentrations of Mg p , Na p , Cl p , and Zn p (Figures 1(b), 1(c), and 1(d)) in the plasma were detected.

Excretion of Urinary Elements.
In the SMV-intoxicated animals, Mg u , Ca u , and V u were excreted in urine in higher amounts than those found in the control rats ( Figures  2(c), 2(f), 3(c), and 3(f)). The urinary Cl u excretion (per Cre u /24 h) also tended to be higher, compared with the control (Figure 3(e)). In turn, the urinary excretion of Zn u (per 24 h and per Cre u /24 h) distinctly decreased in the SMV-exposed rats, in comparison with the control animals (Figures 2(d) and 3(d)), whereas the urinary K u excretion (per 24 h, Figure 2(e), and per Cre u /24 h, Figure 3(e)) dropped and did not change, respectively. However, the urinary levels of Cu u and Na u (per 24 h and per Cre u /24 h), in the SMV-exposed rats, did not turn out to be significantly altered, compared with the control animals (Figures 2(d), 2(e), 3(d), and 3(e)).

Concentrations of Elements in the Kidney and FD.
The exposure to SMV elevated the concentrations of Mg k , K k (Figures 4(b) and 4(d)), and V k (Figure 4(e)) and decreased the concentration of Cu k (Figure 4(c)) in the kidney, compared with the control. A clear tendency toward decreased and increased concentrations of Ca k and Zn k , respectively (Figures 4(b) and 4(c)), was also observed in the SMVintoxicated animals, in comparison with the control rats. However, the concentration of Na k in the same organ of the SMV-exposed rats did not change markedly, compared with the control (Figure 4(d)). In turn, the concentrations of Mg FD and Ca FD in FD decreased (Figures 5(a) and 5(b)), whereas the concentration of V FD in the same FD increased ( Figure 5(c)) after the SMV intoxication, in comparison with the control.

Kidney Weight and Renal MDA and TAS Levels.
The SMV-exposed rats had a significantly elevated relative renal weight (RRW) and the renal MDA k level, compared with the control animals (Figure 4(a)). The renal TAS k level in the same group of rats was also distinctly enhanced, in comparison with the control (Figure 4(a)).

Correlations between Some Measured Variables.
Significant positive and negative correlations and trends toward them were revealed between many different parameters, among others, between the renal V k concentration, the renal MDA k , or TAS k levels and some biomarkers and basic indices as well as between both OS markers (Table 3). They were also found between the fluid intake, BW, EUV, or the urinary Cre u excretion and some investigated parameters (Table 3) as well as between the renal V k concentration or the renal MDA k level and the levels/concentrations of elements in the plasma, urine, kidney and FD ( Table 4). The relationships between the urinary excretion of some elements and Cre u or EUV were also indicated (Table 4).

Discussion
To our knowledge, the present report is the first to describe in a rat model the influence of the 12-week SMV exposure on the levels/activities of a wider panel of biomarkers of renal toxicity and on the concentrations of some elements in the kidney. It reveals a probable mechanism of the development of the SMV-induced functional renal disorders and proposes potential biomarkers of nephrotoxicity for specific conditions of SMV intoxication. It also illustrates for the first time many relationships between some explored indices. Because of different experimental conditions used, comparison of the results presented in the current report with literature data was difficult; therefore, the discussion section relies predominantly on our own results. The increase in the kidney/body weight ratio observed by us in the present (Figure 4(a)) and previous study [28] in the SMV-intoxicated rats may indicate that V accumulated sufficiently in the kidney to manifest significant alterations in the size of this organ. Taking into account the fact that one of the main excretory routes for V within the body is the urinary system [29], V accumulation in the kidney may result in not only functional but also structural damage. Therefore, the estimation of the structural renal injury by histological examination in the rats after the 12-week SMV exposure is going to be performed in the nearest future. In this report, the degree of kidney damage has been evaluated only biochemically.
The decreased CreC together with the enhanced plasma Cre p and U p levels (Figure 1(a)), and the lowered urinary Cre u and U u excretion (Figure 2(a)) found in the SMV-exposed rats may indicate a problem with removal of Cre and U Data are presented as the correlation coefficients ( ) and the levels of statistical significance ( ). p,u,k Plasma, urine, and kidney (concentration), respectively. Fluid-I, Food-I, RRW, BW, and EUV: fluid intake, food intake, renal relative weight, body weight, and excreted urinary volume, respectively. † P < 0.01; * P < 0.05; f P = 0.051; † † † P = 0.053; ff P = 0.054; ‡ ‡ P = 0.055; * * P = 0.058; a P = 0.06; c P = 0.08; d P = 0.09; e P = 0.10; f P = 0.11; g P = 0.12; h P = 0.13; i P = 0.14. , Expressed per 24 h and per Cre u per 24 h, respectively. The significant correlations and tendencies toward them are highlighted in normal and italic bold font, respectively.
from the body and implicate glomerular functional disorders reflected by decreased clearance of the above-mentioned compounds. However, the lowered basic parameters such as fluid intake (discussed by us more widely) [30] and BW found in the same group of animals (Table 2) and the significant correlations between them and Cre u , Cre p , CreC, U u , and/or U p (Table 3) do not allow us to exclude the possibility that dehydration and/or low BW affected the measured standard renal biomarkers. However, it is difficult to recognize to what extent they could influence the alterations in the biomarkers examined. The levels of Cre and U in the blood/urine are known to be affected not only by renal but also by some nonrenal factors [31][32][33]. On the other hand, the correlations between the renal V concentration (V k ) and Cre u , Cre p , CreC, U u , and U p (Table 3) confirm the involvement of V in the changes in the basic biomarkers explored. Moreover, the correlations between V k and both OS markers such as MDA k and TAS k as well as between MDA k and Cre u , Cre p , CreC, and U u (Table 3) also point to the involvement of the SMV-induced OS in their alterations observed. Additionally, the positive correlation between both OS markers allows us to definitively conclude that the changes in the renal TAS level (Table 3) are associated with the alterations in LPO in the kidney due to the SMV exposure. The markedly elevated urinary excretion of CysC u (a tubular injury biomarker) [20,32] demonstrated in the SMVintoxicated rats (Figures 2(a) and 3(a)) without significant alterations in the plasma level of this protein (CysC p ) (Figure 1(a)) may point to decreased reabsorption of CysC by injured tubules. Moreover, the positive correlation of CysC u with TAS k as well as the clear trends toward the positive correlations of CysC u with V k and MDA k ( Table 3) can suggest involvement of the renal V prooxidative action in the proximal tubular dysfunction (PTD), which consequently led to the urinary CysC wasting. It has been reported that when the renal proximal tubule remains intact CysC is completely taken up by the proximal tubular cells (PTC) [32], but in the case of tubular disease and the proximal convoluted tubule (PCT) injury the concentration of this protein in the urine is significantly elevated [34,35]. In addition, the markedly enhanced urinary KIM-1 u excretion (Figure 3(a)) found in the same group of rats may also point to damage to PTC due to the SMV exposure. KIM-1 is expressed at very high levels in proximal tubule epithelial cells after toxic injury and it is present in urine when the injury of PTC occurs [20].
In turn, the lowered urinary activities of LDH u , ALP u, GGTP u , and NAG u , demonstrated in the SMV-exposed rats (Figure 2(b)), allow us to suggest that the use of these tests for monitoring kidney injury during the SMV intoxication may not be valid, as the activity of these enzymes in the urine usually rises due to release from damaged cells. The lowered urinary ALP u and GGT u excretion and the simultaneous increase in the urinary LDH u and NAG u excretion were demonstrated by De la Torre et al. [36] in adult rats after vanadate intoxication. The discrepancies between our results concerning the urinary excretion of LDH and NAG and the results obtained by the above-mentioned investigators draw attention to the dependence of the renal effects on the conditions of vanadate exposure. In turn, the significant negative correlations found between the renal V k concentration and the renal LDH k , ALP k , and GGTP k activities (Table 3) may indicate a direct V impact. The direct inhibitory effect of both vanadate and vanadyl on the ALP activity was described by Cortizo et al. [37]. However, the marked increase in the plasma LDH activity (LDH p ) demonstrated in the SMV-exposed rats (Figure 1(a)) requires further analyses. The measurement of the LDH isoenzyme might be the next important point in a more precise explanation of the significant increase in the activity of this enzyme in the plasma because the total plasma LDH is a highly sensitive test but, simultaneously, it is nonspecific. At the present stage of our study, we may only confirm the involvement of V and the SMV-induced OS in the changes in the activity of LDH in the blood, as evidenced by the significant positive correlations of LDH p with V k and MDA k (Table 3). Data are presented as the correlation coefficients ( ) and the levels of statistical significance ( ). p,u,FD,k Plasma, urine, femoral diaphysis, and kidney (concentration), respectively. EUV: excreted urinary volume. † P < 0.01; * P < 0.05; P = 0.053; # P = 0.056; a P = 0.061; b P = 0.08; c P = 0.20. , Expressed per 24 h and per Cre u per 24 h.
The significant correlations and tendencies toward them are highlighted in normal and italic bold font, respectively.
Additionally, the strong positive correlations of the urinary pH with V k and with both OS markers (Table 3) and the correlation between the renal V k and K k concentration and between the renal V k concentration and the plasma K p level (Table 4) also suggest involvement of OS in the mechanism of the rise in urine pH under the conditions of the SMV exposure (Figure 2(a)). They also confirm the dependence between the accumulation of V in the kidney and disorders in K homeostasis (Figures 1(b) and 4(d)). All these results suggest further measurements to elucidate the mechanism(s) of the changes observed in our experimental conditions. The urinary Mg wasting (Figures 2(c) and 3(c)) and its elevated and diminished concentration in the kidney and FD, respectively (Figures 4(b) and 5(a)), as well as the clearly enhanced plasma and urinary Ca level (Figures 1(b) and