Impaired endocytosis in proximal tubule from subchronic exposure to cadmium involves angiotensin II type 1 and cubilin receptors

Abstract

Background: Chronic exposure to low cadmium (Cd) levels produces urinary excretion of low molecular weight proteins, which is considered the critical effect of Cd exposure. However, the mechanisms involved in Cd-induced proteinuria are not entirely clear. Therefore, the present study was designed to evaluate the possible role of megalin and cubilin (important endocytic receptors in proximal tubule cells) and angiotensin II type 1 (AT1) receptor on Cd-induced microalbuminuria.

Methods: Four groups of female Wistar rats were studied. Control (CT) group, vehicle-treated rats; LOS group, rats treated with losartan (an AT1 antagonist) from weeks 5 to 8 (10 mg/kg/day by gavage); Cd group, rats subchronically exposed to Cd (3 mg/kg/day by gavage) during 8 weeks, and Cd + LOS group, rats treated with Cd for 8 weeks and LOS from weeks 5-8. Kidney Cd content, glomerular function (evaluated by creatinine clearance and plasma creatinine), kidney injury and tubular function (evaluated by Kim-1 expression, urinary excretion of N-acetyl-β-D-glucosaminidase (NAG) and glucose, and microalbuminuria), oxidative stress (measured by lipid peroxidation and NAD(P)H oxidase activity), mRNA levels of megalin, expressions of megalin and cubilin (by confocal microscopy) and AT1 receptor (by Western blot), were measured in the different experimental groups. Data were analyzed by one-way ANOVA or Kruskal-Wallis test using GraphPad Prism 5 software (Version 5.00). P < 0.05 was considered statistically significant.

Results: Administration of Cd (Cd and Cd + LOS groups) increased renal Cd content. LOS-treatment decreased Cd-induced microalbuminuria without changes in: plasma creatinine, creatinine clearance, urinary NAG and glucose, oxidative stress, mRNA levels of megalin and cubilin, neither protein expression of megalin nor AT1 receptor, in the different experimental groups studied. However, Cd exposure did induce the expression of the tubular injury marker Kim-1 and decreased cubilin protein levels in proximal tubule cells whereas LOS-treatment restored cubilin levels and suppressed Kim-1 expression.

Conclusion: LOS treatment decreased microalbuminuria induced by Cd apparently through a cubilin receptor-dependent mechanism but independent of megalin.

Figure 1

Effect of subchronic exposure to cadmium on renal function parameters. Urinary flow rate (A), increased in cadmium + losartan (Cd + LOS) group compared to control (CT), losartan (LOS), and cadmium (Cd) groups. Plasma creatinine (B), and creatinine clearance (C) did not change in the all experimental groups studied. Data are means ± SEM, n = 9–10 rats per group. ^aP < 0.05 vs CT, ^bP < 0.05 vs LOS, ^cP < 0.05 vs Cd. Urinary flow rate was analyzed with one way ANOVA and Bonferroni’s multiple comparison test; plasma creatinine and creatinine clearance were analyzed with Kruskal-Wallis and Dunn’s multiple comparison test.

Figure 2

Effects of subchronic exposure to cadmium on markers of proximal tubule damage. Urinary glucose (A), N-acetyl-β-D-glucosaminidase (NAG) (B), and microalbuminuria (C) in control (CT), losartan (LOS), cadmium (Cd) and cadmium + losartan (Cd + LOS) groups. Co-treatment with LOS (Cd + LOS group) decreased microalbuminuria induced by cadmium (Cd group). Urinary glucose (A) and NAG (B) did not change in the all experimental groups. Data are means ± SEM. n = 9–10 rats per group. ^aP < 0.05 vs CT, ^bP < 0.05 vs LOS, ^dP < 0.05 vs Cd + LOS. Urinary glucose and microalbuminuria were analyzed with one way ANOVA and Bonferroni’s multiple comparison tests; urinary NAG was analyzed with Kruskal-Wallis and Dunn’s multiple comparison tests.

Figure 3

Effect of subchronic exposure to cadmium on Kim-1 protein expression. Representative confocal images of: Kim-1 with dipeptidyl peptidase 4 (DppD), as tubule proximal marker. No expression of Kim-1 was observed in the control (CT), losartan (LOS), and cadmium + losartan (Cd + LOS) groups, it was observed only in the cadmium group (Cd). n = 3 rats per group.

Figure 4

Effect of subchronic exposure to cadmium on megalin’s mRNA and protein expression. Megalin expression did not change in losartan (LOS), cadmium (Cd) and cadmium + losartan (Cd + LOS) groups compared to control group (CT), as observed by immunofluorescence micrographs (A). Quantification of fluorescence intensity is shown in panel (B). mRNA levels of megalin did not change in the four experimental groups (C). Data are expressed as LN mRNA of means ± SEM, n = 3-10 rats per group. Fluorescence intensity and mRNA were analyzed with one way ANOVA and Bonferroni’s multiple comparison tests.

Figure 5

Effect of subchronic exposure to cadmium on cubilin’s mRNA and protein expression. Cubilin expression did not change in losartan (LOS), and cadmium + losartan (Cd + LOS) groups compared to control group (CT), but was decreased in cadmium (Cd) group as observed by immunofluorescence micrographs (A). Quantification of fluorescence intensity is shown in panel (B). mRNA levels of cubilin did not change in the four experimental groups (C). Data are expressed as LN mRNA of means or fluorescence intensity ± SEM, n = 3-10 rats per group. Statistical analysis was performed with Kruskal-Wallis and Dunn’s multiple comparison tests for fluorescence intensity, and one way ANOVA and Bonferroni’s multiple comparison tests for mRNA levels.

Figure 6

Effect of subchronic exposure to cadmium on angiotensin II type 1 (AT1) receptor expression. AT1 receptor expression did not change in losartan (LOS), cadmium (Cd) and cadmium + losartan (Cd + LOS) groups compared to control group (CT), (A). Densitometry is shown in panel B. Representative image of one of three independent experiments is shown (A). Relative band intensity was normalized with actin level (B). Data are means ± SEM, n = 3. Statistical analysis was performed with Kruskal-Wallis and Dunn’s multiple comparison tests.