Fig 2: Dual staining of AChE and AQP6 in CCDs. A) AChE was localized to the glomerulus (G) and the nerve fibers along the interlobular arterioles (arrows) B) AChE in CCD cells was stained with the modified Karnovsky-Roots method (arrows). C) AQP6 in the mouse renal cortex was stained by immunohistochemistry (arrowheads). AChE and AQP6 staining did not overlap in the CCDs. The arrows and arrowheads indicate identical cells in panels A and B. Bar=20 µm.

Fig 3: Expression of adrenergic receptors in M-1 cells and histological localization of the β2 receptor in the mouse kidney. A) RT-PCR (upper column) of ADRs in M-1 total cellular RNA (cDNA, lane 1) and genomic DNA (gDNA, lane 3). The α2 (second row) and β2 (third row) ADRs were amplified from M-1 total cellular RNA, but the α1 (first row) and β3 (fourth row) ADRs were not. No positive band was observed in RTase (−) negative control lane (lane 2). Immunoblot of the β2 receptor (lower column) shows a specific 60–75 kDa band (arrowhead). B) and C) Immunohistochemistry of the β2 receptor (B) and AQP6 (C) in the mouse renal cortex (arrows indicate the same points on both images). Arrows indicate AQP6-positive, β2-positive cells, and arrowheads indicate AQP6-positive, β2-negative cells in the CCDs. D) and E) Immunohistochemistry of the β2 receptor (D) and AQP6 (E) in the outer medulla. β2 receptor immunoreactivities were observed to the straight segment of proximal tubules, but not in the collecting ducts. Arrowheads (AQP6 positive cells) indicate the same points on both images. G: glomerulus. Bar=20 µm (B and C) and 100 µm (D and E).

Fig 4: Characterization of M-1 cellular expression of aquaporin A) Characterization of AQP6 mRNA and protein expression in M-1 cells by RT-PCR (upper column) and immunoblot (middle column), respectively. An AQP6 specific band (arrowhead) was amplified from M-1 cellular total RNA (lane 1), M-1 cellular genomic DNA (gDNA, lane 3) and normal mouse kidney total RNA (MK cDNA, lane 4). No positive band was observed in RTase (−) negative control lane (lane 2). Immunoblot demonstrating the AQP6 specific band (arrowhead) and glycosylated band (double arrowhead) in total membrane protein extracted from M-1 cells (lane 1) and murine kidney (MK, lane 2). B) Characterization of AQP2 mRNA and protein expression in M-1 cells by RT-PCR (upper column) and immunoblot (middle column), respectively. AQP2 mRNA was not amplified from M-1 cellular total RNA (lane 1), although it was observed in M-1 gDNA (lane 3) and MK cDNA (lane 4). Similarly, AQP2 protein was not observed in total membrane protein isolated from M-1 cells (lane 1), although it was observed in total membrane protein isolated from the MK (lane 2). An arrowhead and a double arrow indicate the unglycosylated and glycosylated bands, respectively. Immunofluorescence showed the M-1 cells were positive to the AQP6 antibody (A, lower column), but negative to AQP2 antibody (B, lower column). Bar=50µm.