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First published online 31 May 2005
doi: 10.1242/jcs.02406

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Overexpression of claudin-7 decreases the paracellular Cl^– conductance and increases the paracellular Na⁺ conductance in LLC-PK1 cells

Department of Anatomy and Cell Biology, East Carolina University, Brody School of Medicine, Greenville, NC 27858, USA

View larger version (155K): [in a new window]   Fig. 1. Localization of claudin-7 in porcine (A) and rat (B) kidney cortex. Frozen sections of kidney cortex were immunostained with anti-claudin-7 antibody and tubular markers Claudin-7 was found to be localized in epithelial cells of the distal and collecting tubules using the distal tubular marker TSC (a and c) and the collecting tubular marker NCX1 (e and f). Because all the antibodies used were rabbit polyclonal antibodies the images in a and c, and in g and i are from adjacent sections (b and d, and h and j are the corresponding phase-contrast images). Images in e and f are from the same section with double immunolabeling. (A) In porcine kidney, claudin-7 is present in the apical cell junctions (arrows in a and e). Claudin-7 was also present in a punctate pattern at the thick ascending limb of Henle (TALH), identified by BSC-1 (asterisks and arrows in g and i). The junctional staining of claudin-7 (indicated by two arrowheads in g) was not in the TALH as it was not co-localized with the BSC-1 staining (two arrowheads in i). L: lumen of the tubule. (B) In rat kidney, Claudin-7 was present in the distal tubule (a), collecting tubule (e) and TALH (g). Arrows in a, e, and g indicated the claudin-7 signal at the apical surface of tubules. Scale bar: 30 µm (A) and 20 µm (B).

View larger version (75K): [in a new window]   Fig. 2. Expression of claudin-7-GFP did not affect localization of endogenous tight junction proteins. LLC-PK1 cells with or without transfection were grown on coverslips and then fixed in 100% methanol. (A) Localization of tight junction proteins in parental LLC-PK1 cells. Immunostainings for claudin-1 (a), -3 (c), -4 (d), -7 (e) and ZO-1 (f) were clearly present at the cell-cell junction while claudin-2 signal (b) was predominantly localized in the nucleus. This claudin-2 antibody readily recognized the porcine species as seen in the tubular staining of the frozen section of porcine kidney (insert in b). (B) Localization of endogenous tight junction proteins in LLC-PK1 cells expressing claudin-7-GFP. Cells were double-labeled with GFP (b,e,h,k,n,q) and anti-claudin antibodies (a,d,g,j,m,p). Claudin-7-GFP was closely co-localized with claudin-1, -3, -4, -7 and ZO-1, but not claudin-2, at the cell-cell junction area. The staining of the nucleus (d) was claudin-2-specific since this signal was completely abolished after pre-incubation of claudin-2 antibody with claudin-2 peptide prior to the antibody staining procedure (insert in d). This preabsorption/specific peptide inhibition step did not affect the GFP signal of claudin-7-GFP (insert in e). Confocal x-z images (c,f,i,l,o,r) were the superimposed images of claudin-1, -2, -3, -4, -7 and ZO-1 with claudin-7-GFP. Co-localization of tight junction proteins and claudin-7-GFP is seen in yellow. Bar: 15 µm.

View larger version (48K): [in a new window]   Fig. 3. Immunoblot analysis of endogenous tight junction proteins in parental, GFP-transfected, and claudin-7-GFP-transfected cells (mixed clones). (A) Parental (P), GFP-transfected (G), and claudin-7-GFP-transfected (7) cells were lysed in RIPA buffer, and a total of 20 µg protein for each lane was loaded onto the SDS-polyacrylamide gel. Membranes were blotted against claudin-1, -2, -3, -4, -7 and ZO-1 antibodies. Actin staining was used as a loading control. (B) Densitometry analysis of protein expression levels. Following immunoblotting, X-ray films were scanned and band images were analyzed. The relative signal intensity of each band was obtained after background subtraction. The band intensity for parental cells was normalized to 1 and set as the reference. Data were collected from three independent clones with high expression levels of GFP and claudin-7-GFP. The average density values for three separate experiments were plotted for each designated claudin protein.

View larger version (15K): [in a new window]   Fig. 4. Claudin-7 overexpression resulted in an increased TER in culture medium and P1 buffer. (A) LLC-PK1 cells were plated on Transwell plates and cultured for 7 days. TER was measured in the culture medium and determined as described in Materials and Methods. Claudin-7-overexpressing cells (2) showed significantly higher TER (P<0.001) than that of control cells (1). (B) TER was measured in P1 buffer containing 140 mM NaCl. Cells overexpressing claudin-7 (2) had a significant increase (P<0.01) in TER compared to that of control cells (1). Data are represented as means ± s.e.m. from at least three mixed clones with 24 monolayers (n=24) for each experiment. Student's t-test was used for statistical analysis.

View larger version (14K): [in a new window]   Fig. 5. Effect of claudin-7 overexpression on TER in modified P1 buffer. (A) TER measurements were performed on LLC-PK1 cells grown on Transwell plates. 140 mM NaCl in P1 buffer was replaced by 140 mM arginine-HCl. In the solution, HCl dissociated into H+ and Cl–, and H+ bound to OH– in the solution to form water at pH 7.3. Without Na+ in the solution, TER was dramatically increased in cells overexpressing claudin-7 (2) compared to that of control cells (P<0.001). (B) The experimental condition was similar to that in A, but 140 mM NaCl was substituted by 140 mM lysine-HCl. A significant increase in TER was also observed in cells overexpressing claudin-7 (2) (P<0.001). (C) In contrast, TER significantly decreased (P<0.01) in claudin-7-overexpressing cells when 140 mM NaCl was replaced with 140 mM sodium aspartate (2). Data were obtained from at least three mixed clones (n=24 for each experiment) and presented as means ± s.e.m.

View larger version (19K): [in a new window]   Fig. 6. Dilution potential measurements revealed that overexpression of claudin-7 decreased paracellular permeability to Cl– and increased paracellular permeability to Na+. (A) LLC-PK1 cells stably transfected with GFP or claudin-7-GFP were grown on Snapwell filters. Once monolayers reached confluence, the filter rings containing cell monolayers were mounted into EasyMount chambers. Both apical and basal chambers were filled with P1 buffer containing 140 mM NaCl. Subsequently, P1 buffer in the basal chamber was replaced by P2 buffer with 70 mM NaCl (1 and 2) or P3 buffer with 35 mM NaCl (3 and 4). Dilution potential values were significantly lower (P<0.001) in claudin-7-overexpressing cells (2 and 4) compared to that of controls (1 and 3). The reduction in dilution potentials was a combined effect of Na+ and Cl–. (B) The experimental procedure was the same as in A except that NaCl in P1, P2 and P3 buffers was replaced by arginine-HCl to remove the contribution of Na+ on the reduction of dilution potentials in cells overexpressing claudin-7. In this case, dilution potential values were reduced 100% (P<0.001) in claudin-7-overexpressing cells (2 and 4) compared to that of controls (1 and 3). (C) NaCl was replaced by lysine-HCl in the P1, P2 and P3 buffers. The percentage decrease in dilution potentials was almost the same as with arginine-HCl (P<0.001). (D) To eliminate the contribution of Cl– to the dilution potential, sodium aspartate was used in all three buffers. In the absence of a Cl– concentration gradient, the dilution potentials in both control and claudin-7-overexpressing cells changed from positive to negative. The dilution potentials did not decrease; instead, they significantly increased (P<0.001) in cells overexpressing claudin-7 (2 and 4) compared to controls (1 and 3). Data are represented as means ± s.e.m. from at least three mixed clones (n=12 for each experiment).

View larger version (22K): [in a new window]   Fig. 7. The correlation between claudin-7-GFP expression levels and changes in TER and dilution potentials. (A) Immunoblot analysis of claudin-7-GFP expression level. Three single clones were selected for each expression group. Lysates with equal total protein concentration were obtained from cells expressing low, medium and high levels of claudin-7-GFP protein. Immunoblots were probed with anti-GFP polyclonal antibody to detect claudin-7-GFP fusion protein (49 kDa) and GFP (27 kDa). Anti-actin staining was used as a loading control. (B) TER was measured, in the culture medium, on triplicate filters of three different clones expressing low, medium and high levels of claudin-7-GFP protein. A dose response increase in TER was correlated with the increasing expression level of claudin-7-GFP. (C) Dilution potential measurements were performed to determine the relationship between the ion selective and claudin-7-GFP expression level. Cells were grown on Snapwell filters for 8 days. Triplicate filters were used for measurement of dilution potentials in modified P buffer containing 140 mM lysine-HCl. When lysine-HCl concentration in the basal chamber was changed from 140 mM to 70 mM (column 1-4) or to 35 mM (column 5-8) while keeping the apical chamber constant at 140 mM, cells expressing low (column 2 and 6), medium (column 3 and 7) and high (column 4 and 8) levels of claudin-7-GFP displayed a progressive decrease in dilution potentials compared to that of control cells (column 1 and 5). (D) The experimental procedure was the same as described in C except that dilution potentials were measured in buffer containing sodium aspartate. Results indicated a dose response increase in dilution potentials from cells expressing increased levels of claudin-7-GFP (low: column 2 and 6; medium: column 3 and 7; high: column 4 and 8) compared to that of controls (column 1 and 5). *The value is significantly different from the control (P<0.05).

View larger version (16K): [in a new window]   Fig. 8. Paracellular flux of neutral molecules. (A) Paracellular urea flux. Cells were grown on Transwell membranes until confluent. 50 mM urea was added to the basal solution. After incubation for 1 hour at 37°C, 300 µl aliquot was removed from the apical solution. Urea concentration was measured by an ACE clinical chemistry analyzer. Data are represented as means ± s.e.m. from three independent clones (n=12 for each experiment). Urea flux in claudin-7-overexpressing cells (2) was 120% higher (P<0.001) compared to that of the control cells (1). TER was measured in growth media before and after each paracellular urea flux experiment to confirm that the monolayer remained intact during the flux experiments. (B) Mannitol flux. Control or claudin-7-overexpressing cells were plated on Transwell plates until confluent. The basal culture medium was supplemented with 1 mM cold mannitol. The apical medium was the same as the basal medium except that it contained 1 µCi/ml [3H]mannitol. After 1 hour incubation at 37°C, 100 µl of basal medium were taken and replaced with 100 µl fresh medium containing 1 mM unlabeled mannitol. The radioactivity of the 100 µl samples was measured. Data are means ± s.e.m. from three independent clones (n=12 for each experiment). Mannitol flux in claudin-7-overexpressing cells (2) was significantly higher (P<0.001) compared to controls (1).

View larger version (32K): [in a new window]   Fig. 9. A model of the paracellular channel formed by claudin-7. The extracellular domains of claudin-7 from opposing cells constitute a paracellular channel. The mouth of the channel from both sides is negatively charged, which hinders Cl– entry while allowing Na+ to go through.