Expressions Of TRPC6and BK_Ca Channels In Renal Cortex And Hippocampus Of Mouse During The Development

BackgroudTRPC6, a member of the TRPC family, attracts much attention from the public because of its relationship with the disease. In both the brain and kidney, TRPC6serves a variety of functions.Large-conductance Ca2+-activated K+(BKca) channels are widely distributed in a variety of cells and play a pivotal and specific role in many pathophysiological conditions. However, the function of BKca channels in kidney cortex and hippocampus during the postnatal development has not received attention.TRPC6channel as an important ion channel located in podocytes, plays an essential role in regulating calcium homeostasis of the cell signaling. The podocyte is a remarkable cell type, which encases the capillaries of the kidney glomerulus. Podocytes are of keen interests because of their key roles in kidney development and disease. Because they are specialized, terminally differentiated cells surrounding glomerular capillaries, and are the subject of keen interest because of their key roles in kidney development and disease, more and more researchers detected the ion channels on them, which included TRPC6and BKCa channels. BKCa channels are important ion channels located in podocytes and play the essential role in regulating calcium homeostasis cell signaling.ObjectiveThe aim of the present study was to observe the expression and effects of TRPC6in renal cortex and hippocampus during early postnatal development of the mouse.In this study, in order to elucidate the role of BKCa channels during the development, it is essential to establish the location and quantitation of expression of BKCa-Here we wonder whether TRPC6channels undergo developmental changes during the podocyte differentiation, and whether they contribute to the maturation of podocytes.In this research, we studied the undergoing developmental changes of BKCa channels and their contribution to functional maturation of podocytes.MethodsIn the Part Ⅰ study, Adult C57/BL6mice (20g,8-10weeks old) were purchased from the Experimental Animal Center of the Chinese Academy Medical Sciences. Animals were maintained under standard laboratory conditions under artificial12hours light/12hours dark cycle. Two females were paired with one male (2:1) for a period of4-5days until mating. The day of birth was recorded as P0. The brain and kidney were collected on postnatal day1,3,5,7,14,21,28and49(P1, P3, P5, P7, P14, P21, P28and P49). Kidneys and brains were isolated. Immunohistochemistry and Western blotting were used to detect the expression of TRPC6and BKca channels in the mouse kidney and hippocampus of P1, P3, P5, P7, P14,P21,P28and P49.In the Part Ⅱ study, conditionally immortalized mouse podocyte cell lines was cultured at33℃in RPMI1640medium containing10%fetal bovine serum,100U/ml of penicillin-streptomycin and10U/ml of recombinant mouse y-interferon. To induce differentiation, podocytes were cultured at37℃without y-interferon. All studies were performed on undifferentiated cultured podocytes (d0), differentiating podocytes (days3,6,9,12for cells grown under restrictive conditions) and mature podocytes. Using morphological and immunohistochemical techniques, we investigated the development of distribution and expression of TRPC6and BKca channels in conditionally immortalized mouse podocyte cell line.ResultsThe expression of TRPC6was increased in the mouse hippocampus, and there was a significant increase between P7and P14during the postnatal development. Meanwhile, the expression of TRPC6was also detected in glomerulus and tubules, and a decreased expression was found during postnatal maturation of mouse renal cortex.Our results showed that expressions of BKCa channels were found in tubules and corpuscles at all time points. The expression was also observed in all developmental stages of renal corpuscles, such as comma-shaped body, S-shaped body, renal corpuscles of stage Ⅲ and renal corpuscles of stage Ⅳ. During the development, the expression of BKca channels was decreased and the most prominent change of BKca protein level appeared between P7and P14. In contrast, BKca channels were expressed in all regions of the hippocampus at every time points with the level increasing during the development, and there was an abrupt increase between P7and P14.Our results showed that the distribution of TRPC6channels changed with the maturity of podocyte differentiation. The fluorescent intensity of TRPC6on cell surface increased.Our results showed that the distribution of BKCa channels changed with the maturity of differentiation in a conditionally immortalized mouse podocyte cell line. Additionally, the increase of BKca channel protein expression was detected by immunofluorescence staining with confocal microscopy in podocytes, which was consistent with the increase in the density of BKCa channels examined by western blot technique.ConclusionFrom these in vivo experiments, we concluded that the expression of TRPC6was active in the developing mouse kidney cortex, and followed a loss of expression with the development of kidney. Meanwhile, an increased expression was found in the hippocampus with the development. Together, these data suggested that the developmental changes in TRPC6expression might be required for proper postnatal kidney cortex development, and played a critical role in the hippocampus during development.The findings of the present study suggest that BKca channels play an important role during the postnatal development, but their function may be different in renal cortex and hippocampus. This formed the basis for understanding the nephrogenesis and neurogenesis in mice and provided a practically useful knowledge to the clinical and related research.The development changes of TRPC6may contribute to the podocyte maturation and the physiological function. These results have implications for the physiology and development of kidney and will serve as a baseline for future studies designed to investigate developmental changes of ion channel expression in podocytes. Our results suggested that the developmental changes of BKCa channels may help podocytes adapt to changes in pressure gradients occurring in physiological conditions. Those findings may have implications for understanding the physiology and development of kidney and will also serve as a baseline for future studies designed to investigate developmental changes of ion channel expression in podocytes.