No abstract

The purpose of this study was to determine the cellular and subcellular localization of aquaporin-8 (AQP8) in rat kidney and other organs by RT-PCR analyses and by immunoblotting and immunohistochemistry using peptide-derived rabbit antibodies to rat AQP8. RT-PCR and Southern blotting revealed the presence of AQP8 mRNA in all kidney zones. LLC-PK(1) cells transfected with a rat AQP8 construct exhibited strong labeling with the affinity-purified antibodies, whereas controls using cells transfected with the vector, but without the insert, were negative. The labeling was almost exclusively associated with intracellular vesicles. Immunoblotting of kidney membrane fractions revealed a predominant single band of 26-28 kDa. AQP8 immunoreactivity was mainly present in the cortex and outer stripe of the outer medulla. Sequential ultracentrifugation of rat kidney membrane revealed that AQP8 resides predominantly in intracellular vesicular fractions. Immunocytochemistry revealed modest labeling of proximal tubules and weak labeling of collecting ducts in cortex and medulla of rat kidney. The labeling was confined to cytoplasmic areas with no labeling of the brush border. Immunoblotting and RT-PCR/Southern blotting also revealed the presence of AQP8 protein and mRNA in rat liver, testis, epididymis, duodenum, jejunum, colon, and bronchi/trachea. Consistent with this, immunohistochemistry revealed AQP8 labeling in the hepatocytes and spermatogenic cells in testis and in the basal cells in ductus epididymis, trachea, and bronchial epithelia. Moreover, AQP8 labeling was observed in the myoepithelial cells in salivary, bronchial, and tracheal glands with no labeling of acini or ductal epithelial cells. AQP8 is also present in the surface epithelial cells in duodenum, jejunum, and colon. In conclusion, AQP8 is expressed at low levels in rat kidney proximal tubules and collecting ducts, and it is present in distinct cell types in liver, testis, epididymis, duodenum, jejunum, colon, trachea, and principal bronchi as well as in multiple glands, including salivary glands.

No abstract

The purpose of this study was to examine whether hypokalemia is associated with altered abundance of major renal Na+ transporters that may contribute to the development of urinary concentrating defects. We examined the changes in the abundance of the type 3 Na+/H+ exchanger (NHE3), Na+-K+-ATPase, the bumetanide-sensitive Na+-K+-2Cl− cotransporter (BSC-1), the thiazide-sensitive Na+-Cl− cotransporter (TSC), and epithelial sodium channel (ENaC) subunits in kidneys of hypokalemic rats. Semiquantitative immunoblotting revealed that the abundance of BSC-1 (57%) and TSC (46%) were profoundly decreased in the inner stripe of the outer medulla (ISOM) and cortex/outer stripe of the outer medulla (OSOM), respectively. These findings were confirmed by immunohistochemistry. Moreover, total kidney abundance of all ENaC subunits was significantly reduced in response to the hypokalemia: α-subunit (61%), β-subunit (41%), and γ-subunit (60%), and this was confirmed by immunohistochemistry. In contrast, the renal abundance of NHE3 in hypokalemic rats was dramatically increased in cortex/OSOM (736%) and ISOM (210%). Downregulation of BSC-1, TSC, and ENaC may contribute to the urinary concentrating defect, whereas upregulation of NHE3 may be compensatory to prevent urinary Na+ loss and/or to maintain intracellular pH levels.

The nonerythroid expression of the Duffy blood group protein (gp-Fy) was confined to certain cell types. Immunocytochemistry studies of the kidney showed gp-Fy in the endothelium of glomeruli, peritubular capillaries, vasa recta, and the principal cells (epithelial) of collecting ducts. Gp-Fy was also produced in the endothelial cells of large venules and epithelial cells (type-I) of pulmonary alveoli. In the thyroid, only the endothelial cells of capillaries produced gp-Fy. In the spleen, the endothelial cells of capillaries, high endothelial venule, and sinusoids produced abundant gp-Fy. Ultrastructural studies showed that apical and basolateral plasma membrane domains, including caveolae, had gp-Fy. Immunoblot analysis showed substantially less gp-Fy in nonerythroid cells than in erythrocytes. Moreover, the analyzed nonerythroid organs of Duffy-negative individuals did not produce more gp-Fy to compensate for the lack of this protein in their erythrocytes. The nucleotide sequence and the size of kidney mRNA from a Duffy-positive individual were the same as that of bone marrow. It is assumed, therefore, that nonerythroid Duffy protein is the product of the same gene as that of bone marrow. This notion is reinforced by the fact that nonerythroid and erythroid gp-Fy have the same antigenic domains.