Expression of Aquaporin-6 in Intercalated Cell subtypes in Rat and Mouse Kidney / 대한해부학회지

Aquaporin-6 (AQP6) is a water channel protein located in intracellular vesicles of the proximal tubules and in intercalated cells (ICs) in the collecting duct (CD) of the rat kidney. The function of AQP6 is unknown. However, it colocalizes with vacuolar H+-ATPase in type-A ICs, indicating that it may be important for the function of proton pumps in these cells. The aims of this study were to compare the expression of AQP6 between rat and mouse kidneys, and to establish which types of IC express AQP6. Kidneys of adult male rats and mice were processed for immunohistochemistry using antibodies against AQP6, H+-ATPase, and anion exchanger 1 (AE1). AQP6 was expressed in the S1, S2, and S3 segments of the proximal tubule and in ICs of the CD and connecting tubule (CNT) in both rats and mice. In the rat proximal tubule, AQP6 immunoreactivity was present in intracellular vesicles, whereas in the mouse proximal tubule it was present in the brush border as well as in intracellular vesicles. Triple immunostaining for AQP6, AE1, and H+-ATPase revealed that AQP6 was expressed only in type-A ICs in the CDs and CNTs of both rats and mice, and that the staining was diffuse throughout. There was no AQP6 labeling of type-B ICs, non-A-non-B ICs, or principal cells. The functional significance of the expression of AQP6 in proximal tubule cells and type-A ICs remains to be established. We propose that AQP6 is involved in the retrieval and maintenance of H+-ATPasecontaining vesicles in acid-secreting epithelial cells in the kidney.

Expression of Aquaporin-3 and Aquaporin-4 in Aquaporin-1 Knockout Mice / 대한해부학회지

Recent studies demonstrated impaired urinary concentrating ability in AQP1 knockout mice. To establish of the lack of AQP1 was associated with compensatory changes in other aquaporins, we examined the expression of AQP3 and AQP4 in AQP1 knockout mice. In AQP1 (+/+) mice, there was strong basolateral AQP3 immunostaining in principal cells throughout the collecting duct and strong basolateral AQP4 immunostaining in IMCD cells and principal cells in the medulla. In AQP1 (-/-) mice, there was an increase in AQP3 immunostaining in principal cells in cortex and outer medulla, but no changes in cellular distribution of labeling. In contrast, AQP4 immunostaining was slightly decreased and there was a surprising decrease in cell height and disappearance of immunolabeling of the lateral cell membrane in IMCD cells with only basal labeling remaining. Immunnoblot analysis confirmed the increase in AQP3 expression in cortex (259+/-50%, P<.0.01), outer medulla (607+/-115%, P<0.01) and inner medulla (1,289+/-174%, P<0.0001), and the decrease in AQP4 expression in the inner medulla (31+/-2%, P<0.02) of AQP1 (-/-) compared with AQP1 (+/+) mice (values in AQP1 (-/-) expressed as percentage of AQP1 (+/+)). In summary, AQP1 gene deletion is associated with an upregulation of AQP3 and a downregulation of AQP4. Taken together, these observations suggest that AQP3 and AQP4 are differently regulated and the basolateral water channel expression is upregulated in the cortical and outer medullary collecting duct in AQP1 knockout mice. The significance of the disappererance of AQP4 immunoreactivity in the lateral plasma membrane of IMCD cells in AQP1 knockout mice remains to be established.

Immunocytochemical Study of Calbindin D(28k) Positive Cells in the Developing Rat Kidney / 대한해부학회지

Calbindin D(28k),a calcium binding protein,is found in various tissues,including some cells in the distal nephron.It plays an important role in the regulation of calcium reabsorption. We previously reported the expression of calbindin D(28k) in adult rat kidney.However,the exact time of expression during differentiation in the embryonic kidney is not known.During development,intercalated cells are deleted from the medullary collecting duct by two distinct mechanisms.However,the reason for the different modes of cell death is not known.As calbindin is reported to protect cells against apoptosis,we examined the expression of calbindin D(28k) in the developing rat kidney.Kidneys from 16-,17-,18-and 20-day-old fetuses and 1-,3-,5-,7-,14-and 21-day-old pups and adult Sprague awley rats were processed for immunohistochemistry using a monoclonal antibody against calbindin D(28k) .Intercalated cells were identified by immunostaining for H+ -ATPase and by electron microscopy.Calbindin D(28k) immunoreactivity first appeared in subpopulations of cells in the connecting tubule and medullary collecting duct in the 17-day-old fetus.In the connecting tubule,calbindin D(28k) was expressed only in H+ -ATPase negative connecting tubule cells,and there was no labeling of intercalated cells.In the medullary collecting duct,calbindin D(28k) immunostaining was observed in a few cells with apical H+ -ATPase,characteristic of type A intercalated cells.The numbers of calbindin D(28k) -positive type A intercalated cells increased from day 18 of gestation.In contrast,there was little or no calbindin D(28k) immunoreactivity in the type B intercalated cells or principal cells.During the first two weeks after birth,calbindin D(28k) -positive type A intercalated cells were lost from the terminal part of the medullary collecting duct by simple extrusion. After two weeks,calbindin D(28k) immunostaining decreased in the type A intercalated cells throughout the medullary collecting duct.However,the immunoreactivity of calbindin D(28k) in the cortical collecting duct was increased in some of the type A intercalated cells and the adult pattern was observed in 21-day-old pups.Thus,we propose that the different expression of calbindin D(28k) in type A and type B intercalated cells may be responsible-at least partly-for the different modes of cell death demonstrated in these cells during kidney development.

Immunolocalization of Protein Kinase C Isoenzymes alpha, betaI, betaII and gamma in Adult and Developing Rat Kidney

Protein kinase C (PKC) plays an important role not only in signal transduction mechanisms in various biological processes, but also in the regulation of growth and differentiation during development. We studied the classical PKC alpha, betaI, betaII and gamma, with regard to their expression in adult and developing rat kidney. PKCalpha appeared in the ureteric bud at embryonic day (E) 16, and the proximal and distal anlage at E18. After birth, the immunoreactivity of PKCalpha gradually decreased. In adult, PKCalpha was expressed intensely in the connecting tubule (CNT), the collecting ducts (CD) and the renal corpuscle, and weakly in the proximal and distal tubules. PKCbetaI appeared in the ureteric bud at E16, and the proximal anlage at E18. After birth, the immunoreactivity of PKCbetaI gradually disappeared from the CD and proximal tubule. In adult, PKCbetaI was expressed in the intercalated cells of the CNT and cortical CD, the proximal straight tubule, and the renal corpuscle. PKCbII appeared in distal anlage at E18, and increased markedly after birth. In the CD, PKCbetaII immunoreactivity appeared after birth. In adult, PKCbetaII was expressed in the distal tubule, the CNT and the CD. The immunoreactivity for PKCgamma appeared only in the proximal anlage at E18, and increased temporally around the time of birth. However, no immunoreactivity for PKCgamma was observed in adult rat kidney. These results indicate that classical PKC isoforms appear to play a role in the regulation of various renal functions and differentiation within specific functional units of the uriniferous tubule in rat kidney.