Background: Tight junctions (TJs) are the most apical intercellular junctional complexes in epithelia. These demark apical and basolateral aspects of the plasma membrane, enabling vectoral transport of molecules. Within the epithelial monolayer lining the collecting duct (CD) of the kidney, the tightly regulated transcellular transport of Na+ and H2O critically maintains both the volume and osmolality of our circulating volume. Here, corticosteroid hormones modulate Na+ transport, signalling via mineralocorticoid (MR) and glucocorticoid (GR) receptors. In a model of CD epithelia with deletion of the GR, preliminary electrophysiological measurements demonstrated attenuated transepithelial voltage (Vt) and resistance (Rt). This suggests a role of glucocorticoid signalling in the development and/or maintenance of TJs in the CD, though the specific TJ proteins involved remains unclear.
Aim: To assess the role of glucocorticoid signalling in the development of polarised and electrically resistive CD epithelia and determine which TJ proteins are involved.
Methods: Two experimental models were employed. Firstly, comparison of mCCDcl1 murine CD cells [1] with Nr3c1 deletion (GR knockout, GR-KO) with non-targeting controls (SHAM). Secondly, parental mCCDcl1 cells grown in culture media with (DEX) or without (NO-DEX) the glucocorticoid dexamethasone. All cells were cultured on permeable supports for 9-11d, Vt and Rt were measured by epithelial volt-ohm-meter. RNA was extracted and expression of genes encoding TJ protein families: Zona occludens, Occludin, Claudins, Tricellulin and Angulins were quantified by qRT-PCR. SHAM and GR-KO cells on filters were labelled with antibodies against: GR, ZO-1, Claudin7 and Claudin8, and imaged using a Zeiss AxioScanZ1 slidescanner. Data are shown as mean±SD and statistical significance determined by Mann-Whitney or unpaired t-test, where appropriate.
Results: In GR-KO cells, Rt was negligible compared to SHAM cells after 9d, measuring 0.3±0.1 kΩ·cm2 and 5.6±0.4 kΩ·cm2, respectively (n=8, p<0.001). Similarly, Vt was greatly reduced compared to SHAM cells at 9d, measuring ‑0.9±0.6 mV and -28.0±11.1 mV, respectively (n=8, p<0.001). Interestingly, Rt remained unaltered in NO-DEX cells compared to DEX cells after 9d, measuring 2.5±0.5 kΩ·cm2 and 2.3±0.3 kΩ·cm2, respectively (n=6). Vt, however, was greatly reduced in NO-DEX cells compared to DEX cells after 9d, measuring -14.9±3.4 mV and -50.3±7.2 mV, respectively (n=6, p<0.01).
Across the panel of genes assessed, GR-KO cells had decreased Tjp3, Cldn3, Cldn7, Cldn8 and Ildr1 (n=8, p<0.05), but increased Cldn1, Cldn2 and Cldnd1 (n=8, p<0.01). NO-DEX cells exhibited decreased expression of Tjp1, Tjp2, Tjp3, Cldn8, Cldn12 and Ildr1 (n=8, p<0.05), but increased expression of Ocln, Cldn2 and Cldnd1 (n=8, p<0.01), compared to DEX cells.
Qualitative assessment of immunolabelled cells confirmed loss of nuclear GR but no change to lateral ZO-1 labelling in GR-KO cells (n=6). Furthermore, initial studies revealed lateral labelling of Claudin7 and 8 in SHAM cells was greatly reduced in GR-KO cells (n=2).
Conclusions: Together these data provide evidence that glucocorticoid signalling plays an important role in the development of polarised, transporting CD epithelia. Whilst a number of TJ genes were modified by loss of receptor or removal of ligand, Claudin7 and Claudin8 protein expression was modified, indicating their possible role in development/maintenance of Vt and Rt.