Figures & data
Figure 1 Detection of connexin 30.3 (Cx30.3) mRNA (A) and protein (B) in the kidney. (A) mRNA isolated from mouse whole kidney (lane 1) or from a mouse renal collecting duct cell line (M1, lanes 3, 5) was amplified by RT-PCR to detect Cx30.3 and β-actin (lanes 2, 4, 6). Bands approximately at the predicted sizes of 672 and 350 bp, respectively, were observed in both samples. (B) Western blotting with Cx30.3 antibodies produced a band of approximately 37 kDa in wild-type (WT) mouse kidney homogenates, while tissue homogenates from Cx30.3 knockout mice (KO; n = 4 each) failed to produce a signal. Incubation with a β-actin antibody produced a band of approximately 42 kDa and confirmed equal protein loading amounts. (C) Western blotting with Cx30.3 antibodies produced a single band of approximately 37 kDa in wild-type mouse, rat and rabbit kidney homogenates (n = 2 each).
Figure 2 Renal Cx30.3 expression analyzed by staining for nuclear lacZ reporter gene expression (blue) in heterozygous Cx30.3+/lacZ adult mouse kidneys (A, C–E). (A, B) Cross section through the whole kidney. (A) The most intense lacZ staining was found in the inner medulla (IM). Labeling was weak in the outer medulla (OM) while the renal cortex (C) appeared to be devoid of lacZ staining. (B, F) Wild-type kidney samples served as negative controls. (C–F) High magnification of the IM (C), OM (D, F), and C (E) kidney regions. (C) In the IM, cell nuclei of thin tubular structures stained positive, whereas cells of the large collecting ducts (*) were not labeled. (D) In the OM, only a few tubular structures showed lacZ staining (*), but the dominant structure of the region, the medullary thick ascending limbs (mTAL) were not labeled. (E) In the cortex, only select cells of branching tubular structures, reminiscent of the collecting duct (*) were positive for lacZ. Other tubules, the vasculature and glomeruli (G) were negative. (F) High magnification of the OM region in a wild-type kidney shows no labeling. Bars: 100 μ m.
Figure 3 Detection of Cx30.3 in the mouse kidney by immunofluorescence (red). (A, B, D) Specific staining was observed in wild-type mouse sections labeled with Cx30.3 antibodies. (C) Labeling of Cx30.3 knockout mouse tissue failed to produce a signal. Mostly punctate and cytosolic labeling was observed in only a few cells in the renal cortex, in select cells of the cortical collecting duct (CCD) (A). Occasionally, some staining resembling apical membrane localization was also observed (B). Other cortical structures including the proximal tubule (PT) were devoid of staining. In the renal medulla, dense labeling of thin-walled tubular structures was found. The Cx30.3-positive tubules (*) did not label with AQP1 (green), a marker of the descending thin limb of the loop of Henle (#). Nuclei are stained with 4′,6′-diamidino-2-phenylindole (DAPI; blue). Bars: 10 μ m.
Figure 4 Immunofluorescence labeling of Cx30.3 in rat (A, B) and rabbit (C, D) kidney sections. In the rat cortex (A), apical membrane localization was apparent in select cells of the cortical collecting duct (CCD). No other cortical structures were labeled including the proximal tubule (PT). (B) In the rat outer medulla, the Cx30.3-positive cells of the CCD (arrow) did not label with AQP2 (green, arrowheads), a marker of the principal cells of the CCD. Fluorescent labeling was highest in the inner medulla, and localized in thin-walled tubular structures in the loop of Henle (C). Higher magnification with DIC overlay revealed apical membrane labeling in these tubular structures (*) (D). Most parts of the large medullary collecting ducts (#) were devoid of staining (C, D). Nuclei are stained with DAPI (blue). Bars: 10 μ m.
Figure 5 Schematic representation of Cx30.3-expressing nephron segments (bold line) in both the mouse, rat, and rabbit kidney. G: glomerulus; PT: proximal tubule; DTL: descending thin limb; ATL: ascending thin limb; TAL: thick ascending limb of the loop of Henle; DT: distal tubule; CNT: connecting tubule; CCD: cortical collecting duct; MCD: medullary collecting duct. Cx30.3 labeling was found in the ATL and in select (intercalated) cells of the CCD and MCD.
Figure 6 Immunoblotting analysis of Cx30.3 expression in the rat kidney under various dietary salt conditions. (A) Representative Cx30.3 blots of rat cortical tissue from animals fed a control, high, or low salt diet (n = 4 rats per experimental group). (B) Blots were probed with β-actin to demonstrate even loading. Specific bands for Cx30.3 and β-actin were detected around 37 and 42 kDa, respectively. (C) Densitometric analysis of Cx30.3 expression. No significant difference between control and high salt (p = 0.92) or control and low salt groups was observed (p = 0.09). Shown is mean ± SE of four rats per experimental group.
Figure 7 Immunoblotting analysis of Cx30.3 expression in the kidneys of normotensive (Sprague-Dawley, control) and hypertensive (SHR) rats. (A) A representative blot of control and SHR samples probed with Cx30.3. (B) The same blot probed with villin antibodies to demonstrate even loading. Specific bands for Cx30.3 and villin were detected around 37 and 92 kDa, respectively. (C) Densitometric analysis of Cx30.3 expression. No statistically significant difference in Cx30.3 expression was observed between the two groups (p = 0.85). Shown is mean ± SE of six rats per experimental group.
