Figure 5. Hepsin is the protease mediating uromodulin polymerisation in MDCK cells.

(A) Confocal immunofluorescence analysis showing uromodulin (green), hepsin or prostasin (red) and E-cadherin (blue) (basolateral membrane marker) in polarised MDCK cells, as indicated. Upper panels represent the reconstruction on the xz axis of merged xy scans, for which a representative image is shown in lower panels. Both serine proteases co-localise with uromodulin on the apical plasma membrane of polarised MDCK cells. z stacks = 0.3 µm. A: apical, BL: basolateral. Scale bars, 5 µm. (B) Transcript levels of HPN and PRSS8, as assessed by Real-Time qPCR in MDCK cells transfected with shRNA vectors, as indicated. Expression values (normalised to glyceraldehyde-3-phosphate dehydrogenase, GAPDH) are shown as relative to cells transfected with control vector. Expression of the proteases is specifically reduced in silenced cells. Bars indicate average ± s.e.m. **p<0.01, ***p<0.001 (Student’s t test). The graph represents mean ratios of 3 independent experiments (Figure 5—source data 1). (C) Immunofluorescence analysis showing uromodulin on the surface of MDCK cells transfected with control vector or with shRNA vectors targeting hepsin or prostasin, as indicated. Scale bar, 50 µm. Quantification of the average surface of uromodulin polymers shows that silencing of hepsin, but not of prostastin, substantially reduces uromodulin polymerisation on the membrane of MDCK cells. Bars indicate average ± s.e.m. ***p<0.001 (Mann-Whitney test). The graph represents mean ratios of 3 independent experiments (Figure 5—source data 2).

DOI: http://dx.doi.org/10.7554/eLife.08887.012