Kidney injury molecule-1 is an early biomarker of cadmium nephrotoxicity

Abstract

Cadmium (Cd) exposure results in injury to the proximal tubule characterized by polyuria and proteinuria. Kidney injury molecule-1 (Kim-1) is a transmembrane glycoprotein not normally detected in the mature kidney, but is upregulated and shed into the urine following nephrotoxic injury. In this study, we determine if Kim-1 might be a useful early biomarker of Cd nephrotoxicity. Male Sprague-Dawley rats were given daily injections of Cd for up to 12 weeks. Weekly urine samples were analyzed for Kim-1, protein, creatinine, metallothionein, and Clara cell protein CC-16. Significant levels of Kim-1 were detected in the urine by 6 weeks and continued to increase throughout the treatment period. This appearance of Kim-1 occurred 4-5 weeks before the onset of proteinuria, and 1-3 weeks before the appearance of metallothionein and CC-16. Higher doses of Cd gave rise to higher Kim-1 excretion. Reverse transcriptase-polymerase chain reaction (RT-PCR) expression analysis showed that Kim-1 transcript levels were increased after 6 weeks at the low dose of Cd. Immunohistochemical analysis showed that Kim-1 was present in proximal tubule cells of the Cd-treated rats. Our results suggest that Kim-1 may be a useful biomarker of early stages of Cd-induced proximal tubule injury.

Figure 1. Effects of Cd on body weight, urine volume, urinary creatinine, and urinary protein

Male Sprague-Dawley rats received daily subcutaneous injections of Cd (0.6 mg/kg) for up to 12 weeks, and one day each week, (a) animals were weighed, (b) 24-h urine samples were collected and (c) analyzed for creatinine and (d) protein as described in the Materials and Methods section. Values represent the mean±s.e.m. An * indicates significant differences from week matched control values as determined by two-way ANOVA (P<0.05) and Tukey’s post hoc test. ^#Significant differences from week matched control values as determined by the non-parametric Kruskal-Wallis test (P<0.05) and Dunn’s post hoc test for multiple comparisons; n=10-17 for each data point.

Figure 2. Effects of Cd on the urinary excretion of Kim-1, CC-16, and metallothionein

Animals were treated with Cd (0.6 mg/kg, 5 days per week) for up to 12 weeks and weekly urine samples were analyzed for levels of Kim-1, CC-16, and metallothionein as described in the Materials and Methods section. Values represent the mean±s.e.m. An * indicates significant difference from week matched control values (P<0.05) as determined by two-way ANOVA and Tukey’s post hoc test. ^#Significant difference from week matched control values (P<0.05) as determined by the non-parametric Kruskal-Wallis test and Dunn’s post hoc test for multiple comparisons. For the Kim-1 data, n=16-17 for weeks 1-6, and 10-11 for weeks 7-12; for CC-16, n=10-11; and for metallothionein, n = 5.

Figure 3. Dose dependence of the Cd-induced increase in Kim-1 excretion

Animals were treated with varying doses of Cd (0, 0.6, 1.2, or 2.4 mg/kg/5 days per week for 4 weeks) and urine was analyzed for levels of Kim-1 as described in the Materials and Methods section. The results represent the mean±s.e.m. of six replicate samples for each treatment group. An * denotes significant differences from control values (P<0.05) as determined by the non-parametric Kruskal-Wallis test and Dunn’s post hoc test.

Figure 4. Effects of Cd on the general morphology and the expression of Kim-1 in renal cortex

Rats were treated with Cd (0.6 mg/kg/5 days per week) for 6 or 12 weeks, and representative sections of the renal cortex were processed for hematoxylin and eosin staining and the visualization of Kim-1 (original magnification × 164).

Figure 5. Real time RT-PCR analysis of Kim-1 expression

Total RNA was isolated from the renal cortex and subjected to real time RT-PCR analysis as described in the Materials and Methods section (n=4 for the 6-week controls and 6 for all other treatment groups). An * denotes significant differences from week matched control values as determined by two-tailed t-tests (P<0.05).

Figure 6. The effects of Cd and Hg on renal cell membrane integrity

Animals were treated with either Cd (0.6 mg/kg, subcutaneously 5 days a week for 6 weeks) or HgCl₂ (3.5 mg/kg of Hg, intraperitoneally), and the left kidneys were perfused with ethidium homodimer. Cryosections of the kidneys were then fixed, permeabilized, and labeled with DAPI to identify total nuclei. (a-d) Phase-contrast images corresponding to DAPI-labeled panels (e-h) and ethidium homodimer-labeled panels (i-l). No differences in ethidium homodimer labeling were detected in (j) 6-week Cd²⁺-treated samples or (k) 12-week Cd-treated samples compared with (i)12 week saline-treated control. In contrast, samples from Hg-treated animals showed widespread necrosis (l), as indicated by intense ethidium labeling (original magnification × 164).