Table 1.

Effects of Cd on the localization of cystatin C and megalin in the proximal tubule

Sample	Total labeled pixels/tubule	Total labeled pixels on apical surface	Percent of labeled pixels on apical surface
Cystatin C
Control	94,767 ± 9490	12,515 ± 3026	12.1 ± 2.1
Cd 6 weeks	72,159 ± 5954	26,355 ± 3960	35.7 ± 4.1*
Cd 9 weeks	127,880 ± 27,992	107,114 ± 23,663	76.1 ± 2.2*
Cd 12 weeks	153,179 ± 12,307	136,144 ± 10,923	89.0 ± 2.0*
Megalin
Control	166,802 ± 25,904	45,788 ± 6808	30.3 ± 3.1
Cd 6 weeks	115,589 ± 15,109	48,167 ± 6565	41.2 ± 1.0*
Cd 9 weeks	98,532 ± 13,923	51,438 ± 6580	52.9 ± 1.8*
Cd 12 weeks	124,177 ± 9758	83,655 ± 7.384	67.1 ± 1.8*

The localization of cystatin C and megalin in the peroxidase-labeled kidney sections from Fig. 5 were quantified using the Image J computer program. Using the hematoxylin staining as a morphologic guide, regions of interest were created around each tubule. The number of pixels that were positive for cystatin C or megalin staining was measured using constant thresholding tools (as described in “Materials and methods” section) to identify peroxidase signal above background levels for each tubule. A second region of interest (apical compartment) for each tubule was created to encompass the luminal space and 1–2 cells of the apical luminal surface. Positive cystatin C and megalin pixels were again measured in the apical compartment using the same thresholding parameters. The percentile data of pixels in the apical compartment was calculated and the data were evaluated by a one-way ANOVA with post hoc Tukey’s test. Values represent the mean ± SEM of data from 7 to 10 tubules per image. An asterisks denotes that the percent of pixels in the apical compartment is significantly greater (p < 0.05) than in the control samples