Expressions Of Calbindin-D28k During Mouse Kidney Development

Objective: To observe the expression rules and sites of Calbindin-D28k during kidney development of mouse, and discuss its role in kidney development.Methods: Healthy adult kunming mice of 25-30g were selected and determined whether they were pregnant by examining the vaginal plugs. The mice of embryonic day(E)10,12,14,16,18 and neonatal day (N)1,3,7,14,21,40 were selected with eight mice in each group. The normal developmental process of mouse kidney was studied microscopically. The expression of Calbindin-D28k was examined by Immunohistochemistry staining and immunofluorescence technique and Western blotting. And the content of Calbindin-D28k was analyzed by computerized densitometry.Results:①Microscopy examination: Ureteric buds surrounded by metanephrogenic tissues were observed microscopically at E12. Comma-shaped bodies and S-shaped bodies emerged at E14, and capillary tuft stage (III), immature (IV) bodies emerged at E16. Renal medulla was observed at E18. Renal papillae were seen at N1 and renal corpuscles became matured, ureteric buds disappeared, ray medullary emerged at N7. Cortex became thick at N14. Cortex became thicker, and inner zone of renal medulla were in the wall of renal papillae at N21.Kidney had the same structures as those of adult mice.②Immunohistochemistry examination: Calbindin-D28k expression could not be found in the kidney at E10. At E12, Calbindin-D28k was weakly expressed in the ureteric buds and not expressed in renal metanephrogenic tissues. At E14, Calbindin-D28k expressions were examined in the branches of renal ureteric buds. At E16, the strong expression of Calbindin-D28k was found in the ampullae of ureteric buds and weakly expression in connecting segment of the nephron that link to the ampullary ureteric bud tip and no expression in nephric tubule and S-shaped bodies. From E18 to N7, the strong expression of Calbindin-D28k was found in distal nephron and no expression in renal capsules, proximal tubules and medullary loops. Calbindin-D28k expressions were located in distal convoluted tubules (DCTs), connecting tubules (CNTs), and cortical collecting ducts (CCDs) at N7.The expressions of Calbindin-D28k were decreased at N14,N21,N40.③Computerized densitometry examination: The gray scale value of Calbindin-D28k expression was highest at E12 by CLAS-1000 cellimage analytical system. It became low from E12 to N7 and became high from N14 to N40.④Immunofluorescence examination: At E10, Calbindin-D28k expression could not be found in the kidney. At E12, Calbindin-D28k was weakly expressed in the ureteric buds of kidney. At E14, Calbindin-D28k expressions could be observed in the Y-shaped bodies of ureteric buds of kidney. At E16, the strong expression of Calbindin-D28k was found in the ampullae of ureteric buds and weakly expression in connecting segment of the nephron that link to the ampullary ureteric bud tip. From E18 to N7, the expression of Calbindin-D28k was increased and located in DCTs,CNTs and CCDs. From N7 to N40, the expression of Calbindin-D28k was abundant in the DCTs,CNTs and CCDs , but was decreased in medullary collecting duct. It was localized in the cytoplasm of epithelial cell of ureteric buds or DCTs,CNTs and CCDs.⑤Western blotting examination: At E12, Calbindin-D28k expression could be hardly detected in the kidney. From E14 to E16, the expression was increased [(12.0994±0.6911),(13.2383±0.1729), P<0.01].There was no significant difference from E16 to E18. From E18 to N7, the expression of Calbindin-D28k was increased obviously [(13.8447±0.2385), ( 15.2718±0.2019 ) ,(16.3208±0.2839), (19.3383±0.4044), P<0.01].From N7 to N14, the expression of Calbindin-D28k was decreased with significant difference [(19.3383±0.4044) , (15.8915±1.0185 ) , P<0.01].There was no significant difference between N14 and 40.Conclusion: The expression of Calbindin-D28k is weak at E12 which is enhanced with time until the peak at N7, and decreased thereafter. The expression of Calbindin-D28k is found in DCTs,CNTs and CCDs. The expression is localized in the cytoplasm of epithelial cell of ureteric buds or DCTs,CNTs and CCDs. Calbindin-D28k is a calcium ferry protein in cytoplasm and is very important to renal calciumreabsorption during the development of kidney.