Figure 1.
Human Kidney Epithelial Cells (HK-2) in culture, exposed to Sodium Oxalate showed increased KIM-1 mRNA and protein levels.
(a) Oxalate was added to serum starved HK-2 cells and incubated for various time points. KIM-1 mRNA levels were estimated by Relative quantitative PCR and (Top Panel) representative gel image is shown. (Lower Panel) Quantification of mRNA levels was performed by band density analysis and represented as a relative intensity compared to control. Each data point represents mean +/− S.D. of two independent PCRs (* indicates p<0.05 compared to control, n = 2). (b) Total protein was extracted as in materials and methods. Western blot analysis was used to detect KIM-1 protein levels in the cells. Representative blots from two individual experiments are shown (Top Panel). Densitometric analysis was used to quantify the KIM-1 protein levels. Each data point represents mean +/− S.D. of two independent gels (*indicates p<0.05 compared to control, n = 2).
Figure 2.
Immunohistochemical analysis of KIM-1 expression in kidney tissue of hyper-oxaluric rats.
(Left Panel) KIM-1 protein expression in kidney tissue was detected using immunohistochemistry. (Right Panel) protein expression was quantified based on color density from five different 1 cm2 regions of the section. Each data point represents mean +/− S.D. of two independent sections (* indicates p<0.05 compared to control, n = 2).
Figure 3.
Hyper-oxaluric rats have increased mRNA and protein levels of KIM-1 in renal tissue.
(a) KIM-1 mRNA levels were detected by relative quantitative RT-PCR in kidney tissue of control and three individual hyper-oxaluric rats. Representative gel images from three individual PCRs are shown (Top Panel). Quantitative representation of relative KIM-1 mRNA expression across the hyper-oxaluric group compared to control is shown in the Bottom panel. Each data point represents mean +/− S.D. of three experiments (*indicates p<0.05 compared to control, n = 3). (b) Representative gel images from three individual western blots are shown (Upper Panel). Quantitative representation of the relative amounts of KIM-1 protein in the kidney tissue is shown in the lower panel. Each data point represents mean +/− S.D. of three blots (*indicates p<0.05 compared to control, n = 3).
Figure 4.
Hyper-oxaluric rats showed crystal deposits of Calcium Oxalate in renal tubules after 3 weeks.
(a) Hematoxylin and Eosin stained paraffin embedded 5 um sections were photographed under polarized light to detect crystal deposits in the tubules. Representative Images captured using a 10× objective magnification are shown. (b) Von Kossa's staining method was used to identify Calcium deposition in tubules of hyper-oxaluric rat kidneys. Digital photo-micrographs were taken with a 10× magnification. Representative images are shown.
Figure 5.
Hyper-oxaluria results in increased shedding of KIM-1 ectodomain in rat urine.
(a) Western blot to detect KIM-1 protein shed in the urine of two individual hyper-oxaluric rats. Representative gel images from three individual western blots are shown (Upper Panel). Quantitative representation of the relative amounts of KIM-1 protein in urine, averaged across the treatment groups, is shown in the lower panel. Each data point represents mean +/− S.D. of three blots (* indicates p<0.05 compared to control, n = 3). (b) Urine Volume (ml) and Creatinine excretion (mg) values (c) Oxalate (mg) and (d) Calcium (mg) over a period of 24 hours. Each data point represents mean +/− S.D. of individual animals in each group (* indicates p<0.05 compared to control).
Table 1.
Sequences of primers used in RT-PCR.