Acute renal failure (ARF) induced by potassium dichromate (K₂Cr₂O₇) has been used as a model to study the pathophysiology of this disease [7,9]. Experimental data show that chromate affects selectively the convoluted section of the proximal tubules [7,12-15] and induces acute necrosis of renal tubules [14,15]. Clinical and experimental renal damage induced by K₂Cr₂O₇ has been associated with oxidative stress [15-20]. In fact, some antioxidants such as ascorbic acid, vitamin E, N-acetyl cysteine, and glutathione prevent the K₂Cr₂O₇ induced renal damage [21-25] whereas the inhibition of glutathione biosynthesis enhances it [22,24]. Evidences suggest that reactive oxygen species (ROS) are involved in Cr(VI)-induced cell injury [16,17,19,26]. Chromium reduction intermediates [Cr(V), Cr(IV), and Cr(III)], which could be generated under physiological conditions, may be toxic as they involve ROS production [27-29]. In vitro chromate reduction via H₂O₂ has been shown to produce hydroxyl radicals via a Fenton-like reaction [27,30-33].

Markers of nephrotoxicity are shown in Figs. ​Figs.22 and ​and3.3. Creatinine clearance decreased on days 1–4 (Fig. ​(Fig.2A),2A), and serum creatinine and BUN increased on days 1–4 and 1–6, respectively (Figs. ​(Figs.2B2B and ​and2C).2C). Urinary excretion of NAG and total protein increased on days 1–4 and 1–6, respectively (Figs. ​(Figs.3A3A and ​and3B).3B). The major damage was observed on days 2–4 (Figs. ​(Figs.22 and ​and3).3). On day 8, all these markers returned to control values indicating that K₂Cr₂O₇-induced ARF is reversible which is consistent with previous observations [10]. These functional findings are in a close agreement with the histological data. Significant structural abnormalities were seen in the kidney cortex since the first day after K₂Cr₂O₇ administration. Specifically, 50% of the epithelium from the proximal convoluted tubules showed cellular swelling, necrosis and partial or complete detachment from the basal membrane. On day 2, when the peak of the tubular damage was observed, 70% of the proximal convoluted tubules was affected (Figs. ​(Figs.4B4B and ​and5).5). On days 3 and 4 proximal convoluted tubules showing the above mentioned abnormalities decreased to 40 and 25%, respectively (Fig. ​(Fig.5).5). On day 4, many tubules also showed numerous cellular debris in their lumen and epithelium regeneration was also seen but it was more evident on days 6 and 8 (data not shown). On days 10 and 12 the kidney histology was almost normal (Fig. ​(Fig.4C),4C), only few tubules were revisted by active regenerative epithelium (small cuboidal cells with big nucleus and occasional mitotic figures). The high levels of serum and kidney chromium correlated with the structural and functional renal alterations on days 1–6. Interestingly, the renal damage disappeared in spite of the kidney still having high levels of chromium on days 8–12. This may be explained by the fact that chromium (VI), which is readily taken up into tissues, is reduced inside the cell to the final stable product chromium (III) [19]. The biological effects of chromium (VI) are generally attributed to cellular uptake, because chromium (VI), in contrast to chromium (III), is easily taken up by cells through the sulfate anion transport system [42,43]. However, once inside, chromium (VI) is reduced through reactive intermediates such as chromium (V) and chromium (IV) to the more stable chromium (III) by cellular reductants including glutathione, vitamins C and B₂, and flavoenzymes [42]. Thus, the formation of chromium (III) or other intermediate oxidation states, in particular chromium (V), is believed to play a role in the biological effect of chromium (VI) compounds. In vitro studies have shown that this reduction process causes the generation of active oxygen species [44] which are involved in renal damage [15,34]. Interestingly, it has been shown that a low dose of K₂Cr₂O₇ (10 mg/Kg) is unable to induce nephrotoxicity suggesting a threshold of this compound to induce renal damage [10]. In addition, it is know that chromium is located in vacuoles inside the proximal tubular cells which may delay the excretion of this metal [13]. In fact it has been shown that chromium remains for a long time in several tissues including kidney [13,40,41] which is consistent with our data.

The histological abnormalities correlated with renal functional alterations (days 1–6) and with the immunohistochemical detection of 3-NT and protein carbonyls. DNPH reacts with free carbonyls forming DNP-derived proteins which can be detected using antibodies against DNP. Carbonyl derivatives are formed by ROS mediated oxidation of side-chains of some amino acid residues and are important detectable markers of oxidative damage to proteins [45]. Kidneys from control non-treated animals did not show or only had slight immunostaining to 3-NT or protein carbonyls (Figs. ​(Figs.4D4D and ​and4G),4G), while kidneys from K₂Cr₂O₇-treated animals on day 2 showed strong 3-NT immunostaining in necrotic and swollen tubular epithelial cells, as well as some mesangial cells (Fig. ​(Fig.4E).4E). This increase was significative (P < 0.0001 vs. day 0) (Table ​(Table1).1). In fact, it was observed from day 1 to day 8 (data not shown). Protein carbonyls immunostaining was also clear in the cytoplasm and nuclei from tubular epithelium and mesangial glomerular cells at the same time points (Fig. ​(Fig.4H4H and data not shown) (P < 0.001 vs. day 0) (Table ​(Table1).1). Then, on days 10 to 12, a striking decrease of 3-NT and DNP immunostaining was seen, being similar to the control non-treated rats (Figs. ​(Figs.4F4F and ​and4I).4I). In previous works, we also have found in K₂Cr₂O₇-treated rats an intense 3-NT immunostaining [15] and an increase in protein carbonyl content [15,34]. Our data confirm that ROS are involved in K₂Cr₂O₇-induced nephrotoxicity [16,17,19,26,44] which has additionally been supported by the protective effect of several antioxidants in this experimental model [21-25].

On the other hand, it was found that urinary excretion of NO₂^-/NO₃^- decreased significantly in K₂Cr₂O₇-treated rats on days 2–6 (Fig. ​(Fig.6).6). Urinary excretion of NO₂^-/NO₃^- is considered as an index of NO production [37] and therefore these data may suggest that NO production is decreased in K₂Cr₂O₇-treated rats. However, the strong nitrosative stress observed in our rats by 3-NT immunostaining, may suggest that the decrease in urinary NO₂^-/NO₃^- excretion could be secondary to the NO consumption by its reaction with superoxide anion to generate peroxynitrite and other reactive nitrogen species involved in protein tyrosine nitration [46]. This is supported by the association between the time course alterations in 3-NT immunostaining (days 1–8) and the decrease in urinary NO₂^-/NO₃^- excretion (days 2–6).

Further we investigated the time response of the renal antioxidant enzymes in K₂Cr₂O₇-treated rats. In control rats, strong CAT immunostaining was seen in the epithelial cells from proximal and distal convoluted tubules (Fig. ​(Fig.7A).7A). From day 2 to day 10 after K₂Cr₂O₇ administration, CAT immunostaining showed striking decrease, particularly in the epithelium from tubules with evident cellular damage (Fig. ​(Fig.7B7B and data not shown). Then, at day 12, when almost normal kidney histology was seen, strong CAT immunostaining was observed in a similar pattern than in the kidney from control animals (Fig. ​(Fig.7C).7C). CAT activity (Fig. ​(Fig.8A)8A) and content (Fig. ​(Fig.8B)8B) decreased on days 2–10 and 3–10, respectively. In control animals the epithelial cells from the proximal and convoluted tubules showed strong immunoreactivity to Cu, Zn-SOD and Mn-SOD (Figs. ​(Figs.7D7D and ​and7G).7G). This pattern of immunostaining did not show evident changes during the time course study (days 1–12) (Figs. ​(Figs.7E,7E, ​,7F,7F, ​,7H,7H, ​,7I,7I, and data not shown); even the damaged swollen epithelial tubular cells exhibited strong immunostaining to both SOD enzymes. These data are in agreement with kidney total SOD activity (Fig. ​(Fig.9)9) and with the protein content of Mn-SOD and Cu, Zn-SOD measured by Western blot which remained unchanged at all time points in K₂Cr₂O₇-treated rats (data not shown). Finally, the activity of GPx and GR decreased on days 3–12 and on days 2–10, respectively (Fig. ​(Fig.10).10). It is very clear from the above data that there was a differential response of the antioxidant enzymes to K₂Cr₂O₇ injection. Interestingly, no enzyme (SOD, CAT, GPx, and GR) was altered on day 1 when the kidney damage was very severe. Surprisingly, SOD activity and content remained essentially unchanged at all time points. This may be a consequence of the fact that both SOD enzymes were immunolocalized even in the damaged epithelial tubular cells. In contrast, the other enzymes decreased to reach their lowest values on day 6 returning then to basal values by day 12 with the exception of GPx which remained low. On days 2–6, there was some degree correlation between CAT, GPx, and GR and the markers of nephrotoxicity. However, from day 8 these enzymes remained low in spite of renal function and structure returned to control. Furthermore, the time course study suggests that the decrease in GPx, GR, and CAT activities may be secondary to the oxidative and nitrosative stress which are evident since day 1. In fact, it has been demonstrated that peroxynitrite impairs GPx activity [47] and superoxide anion inactivates GPx [48] and CAT [49]. Interestingly, GR and CAT remained low until day 10 and GPx remained low until day 12 in absence of oxidative and nitrosative stress. The reason why these enzymes remained low at the end of the study is not clear, however we may speculate that factors other than oxidative/nitrosative stress are involved in the diminution in these enzymes and/or proximal tubules have not reached yet the full capacity to synthesize these enzymes. Therefore, additional studies are required to explain why some antioxidant enzymes remained low on days 10–12 in absence of oxidative/nitrosative stress.

BUN Blood urea nitrogen

CAT Catalase

Cu, Zn-SOD Copper and zinc superoxide dismutase

DNP Dinitrophenol

DNPH 2,4-dinitrophenylhydrazine

ECL Enhanced chemiluminiscence

GPx Glutathione peroxidase

GR Glutathione reductase

H₂O₂ Hydrogen peroxide

K₂Cr₂O₇ Potassium dichromate

Mn-SOD Manganese superoxide dismutase

NADPH Nicotinamide adenine dinucleotide phosphate

NAG N-acetyl-β-D-glucosaminidase

NBT Nitroblue tetrazolium

NO Nitric oxide

NO₂^- Nitrate

NO₃^- Nitrite

3-NT 3-Nitrotyrosine

ROS Reactive oxygen species