Time course study of oxidative and nitrosative stress and antioxidant enzymes in K2Cr2O7-induced nephrotoxicity

Abstract

Background: Potassium dichromate (K2Cr2O7)-induced nephrotoxicity is associated with oxidative and nitrosative stress. In this study we investigated the relation between the time course of the oxidative and nitrosative stress with kidney damage and alterations in the following antioxidant enzymes: Cu, Zn superoxide dismutase (Cu, Zn-SOD), Mn-SOD, glutathione peroxidase (GPx), glutathione reductase (GR), and catalase (CAT).

Methods: Nephrotoxicity was induced in rats by a single injection of K2Cr2O7. Groups of animals were sacrificed on days 1,2,3,4,6,8,10, and 12. Nephrotoxicity was evaluated by histological studies and by measuring creatinine clearance, serum creatinine, blood urea nitrogen (BUN), and urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG) and total protein. Oxidative and nitrosative stress were measured by immunohistochemical localization of protein carbonyls and 3-nitrotyrosine, respectively. Cu, Zn-SOD, Mn-SOD, and CAT were studied by immunohistochemical localization. The activity of total SOD, CAT, GPx, and GR was also measured as well as serum and kidney content of chromium and urinary excretion of NO2 -/NO3-. Data were compared by two-way analysis of variance followed by a post hoc test.

Results: Serum and kidney chromium content increased reaching the highest value on day 1. Nephrotoxicity was made evident by the decrease in creatinine clearance (days 1-4) and by the increase in serum creatinine (days 1-4), BUN (days 1-6), urinary excretion of NAG (days 1-4), and total protein (day 1-6) and by the structural damage to the proximal tubules (days 1-6). Oxidative and nitrosative stress were clearly evident on days 1-8. Urinary excretion of NO2-/NO3- decreased on days 2-6. Mn-SOD and Cu, Zn-SOD, estimated by immunohistochemistry, and total SOD activity remained unchanged. Activity of GPx decreased on days 3-12 and those of GR and CAT on days 2-10. Similar findings were observed by immunohistochemistry of CAT.

Conclusion: These data show the association between oxidative and nitrosative stress with functional and structural renal damage induced by K2Cr2O7. Renal antioxidant enzymes were regulated differentially and were not closely associated with oxidative or nitrosative stress or with kidney damage. In addition, the decrease in the urinary excretion of NO2-/NO3- was associated with the renal nitrosative stress suggesting that nitric oxide was derived to the formation of reactive nitrogen species involved in protein nitration.

Figure 1

Chromium concentration in (A) serum and (B) kidney in control (○) and K₂Cr₂O₇ (●)-treated rats. Data are mean ± SEM. *P < 0.001 vs. control group. n = 3–6.

Figure 2

(A) Creatinine clearance, (B) serum creatinine, and (C) blood urea nitrogen control (○) and K₂Cr₂O₇ (●)-treated rats. Data are mean ± SEM. *P at least <0.05 vs. control group. n = 4–6.

Figure 3

Urinary excretion of (A) NAG and (B) total protein in control (○) and K₂Cr₂O₇ (●)-treated rats. Data are mean ± SEM. *P at least <0.05 vs. control group. n = 4–18.

Figure 4

Representative images of histology (first row) and immunohistochemical detection of 3-NT (second row) and DNP (third row) during the evolution of the kidney damage produced by K₂Cr₂O₇. The study was performed in control rats (day 0) and on days 2 and 12 after a single injection of K₂Cr₂O₇ (15 mg/Kg). (A) Normal kidney histology from control rat (H/E). (B) On day 2, there is extensive tubular damage manifested by swollen and necrotic epithelial cells (H/E). (C) total kidney regeneration has been produced on day 12 post K₂Cr₂O₇ administration (H/E). (D) Normal kidney from control animals shows scarce 3-NT immunoreactivity. (E) On day 2, there is strong 3-NT immunostaining in the epithelium from the convoluted tubules. (F) Slight 3-NT immunostaining is observed in the tubular epithelium on day 12. (G) Normal kidney from control rat shows scarce DNP immunostaining in the tubular epithelium. (H) In contrast, on day 2 there is clear DNP immunostaining in the cytoplasm and nucleus of the tubular epithelial cells and mesangium. (I) Slight DNP immunostaining is observed on day 12. 400 X.

Figure 5

Quantitative histological analysis in control (○) and K₂Cr₂O₇ (●)-treated rats. Data are mean ± SEM. *P < 0.05 vs. control group. n = 20 tubules/rat and 5 rats/group.

Figure 6

Urinary excretion of NO₂^-/NO₃^- in control (○) and K₂Cr₂O₇ (●)-treated rats. Data are mean ± SEM. *P < 0.05 vs. Control group. n = 4–10.

Figure 7

Representative immunohistochemical detection of CAT (first row), Cu, Zn-SOD (second row), and Mn-SOD (third row) during acute renal failure induced by K₂Cr₂O₇. The study was performed in control rats (day 0) and on days 2 and 12 after a single injection of K₂Cr₂O₇ (15 mg/Kg). (A) Normal kidney from control rats shows intense CAT immunoreactivity in the proximal and distal convoluted tubules. (B) In contrast, kidney cortex on day 2 shows evident decrease of CAT immunostaining. (C) Strong CAT immunoreactivity in the tubular epithelium is observed on day 12. (D) Normal kidney from control animals also showed strong Cu, Zn-SOD immunostaining in the cortex convoluted tubules. This strong Cu, Zn-SOD immunoreactivity is also observed on day 2 (E) and on day 12 (F). A similar pattern of Mn-SOD immunostaining is observed in normal kidney from control rat (G), and after 2 (H) and 12 (I) days of K₂Cr₂O₇ injection. A-C and G-I 100X, D-F 400X.

Figure 8

(A) Activity and (B) content of CAT in kidney from control (○) and K₂Cr₂O₇ (●)-treated rats. Data are mean ± SEM. *P at least <0.05 vs. control group. n = 4–6.

Figure 9

Total superoxide dismutase activity in kidney from control (○) and K₂Cr₂O₇ (●)-treated rats. Data are mean ± SEM. n = 3–6.

Figure 10

Activity of (A) glutathione peroxidase and (B) glutathione reductase in kidney from control (○) and K₂Cr₂O₇ (●)-treated rats. Data are mean ± SEM. *P at least <0.05 vs. control group. n = 5–6.