Fig 2: Defective uromodulin urinary secretion in mice lacking hepsin.(A) Transcript level of Hpn, as assessed by Real-Time qPCR on microdissected nephron segments (normalised to Gapdh). Expression of Hpn is detected in proximal straight tubules (PST), thick ascending limb (TAL), proximal convoluted tubules (PCT), distal convoluted tubules (DCT) and, to a lesser extent, in collecting ducts (CD). Minimal expression of the protease is detected in glomeruli (GI). Bars indicate average ± s.e.m. of 3 independent experiments (Figure 6—source data 1). (B) Immunofluorescence analysis of mouse kidney sections showing co-localisation of endogenous hepsin and uromodulin on the apical plasma membrane of TAL epithelial cells. Scale bar, 20 µm. (C) Representative Western blot analysis of urinary uromodulin secretion in Hpn-/- mice or control animals. Urinary protein loading was normalised to urinary creatinine concentration. Densitometric analysis shows reduced uromodulin urinary secretion in animals lacking hepsin (average ± s.d., n = 10/group, Figure 6—source data 2). ***p<0.001 (Student’s t test). (D) Western blot analysis of uromodulin in kidney lysates of Hpn-/- mice or control animals. Beta-actin is shown as a loading control. Densitometric analysis shows accumulation of uromodulin in kidney lysates from Hpn-/- mice (average ± s.d., n = 3/group, Figure 6—source data 3). **p<0.01 (Student’s t test). (E) Representative immunofluorescence analysis showing apical plasma membrane signal of uromodulin in kidney sections from Hpn-/- mice or control animals. Scale bar, 50 µm. (F) Transcript level of Umod, as assessed by Real-Time qPCR on total kidney extracts from Hpn-/- mice or control animals (normalised to Hprt1). Expression of Umod is comparable between the two groups of animals (n = 3/group, Figure 6—source data 4). Bars indicate average ± s.e.m. (Student’s t test).DOI: http://dx.doi.org/10.7554/eLife.08887.01510.7554/eLife.08887.016Figure 6—source data 1.Transcript level of Hpn in mouse microdissected nephron segments (Figure 6A).DOI: http://dx.doi.org/10.7554/eLife.08887.01610.7554/eLife.08887.017Figure 6—source data 2.Quantification of urinary uromodulin secretion in Hpn-/- and control mice (Figure 6C).DOI: http://dx.doi.org/10.7554/eLife.08887.01710.7554/eLife.08887.018Figure 6—source data 3.Quantification of uromodulin levels in kidney lysates of Hpn-/- and control mice (Figure 6D).DOI: http://dx.doi.org/10.7554/eLife.08887.01810.7554/eLife.08887.019Figure 6—source data 4.Transcript levels of Umod in Hpn-/- and control mice (Figure 6F).DOI: http://dx.doi.org/10.7554/eLife.08887.019

Fig 3: Altered uromodulin processing and NKCC2 activation in Hlb320 mTAL cells. (a) Representative immunofluorescence analysis for uromodulin (green) in permeabilized mTAL cells derived from Hlb320 and WT mice. A view of the apical cell surface and through the nuclear plan (intracellular), with the z-plan reconstructions, are shown. Nuclei are stained in blue with DAPI. Scale bar: 25 µm. (b) Representative output picture and result of polymer number per field quantification using ImageJ Ridge Detection in mTAL cells from Hlb320 and WT mice. N = 15 fields from 3 mTAL cultures. Scale bar: 50 µm; bars indicate mean ± s.e.m.; ****p = 0.0001 (Unpaired two-tailed t test). (c) Representative immunofluorescence analysis for uromodulin (green) and ER chaperone calnexin (red) in permeabilized mTAL cells derived from Hlb320 and WT mice. Inset: zoom on single cell. Nuclei are stained in blue with DAPI. Scale bar: 50 µm. (d) Representative western blot analysis of uromodulin secreted in the apical compartment of mTAL cells derived from Hlb320 and WT mice. Same apical volumes were loaded in each lane. Densitometric analysis is shown next to the blot. Bars indicate mean ± s.e.m.; ****p = 0.0001 (Unpaired two-tailed t test). (e) Western blot analysis for phosphorylated (Thr96 and Thr101) and total NKCC2 in mTAL cells from Hlb320 and WT mice. ß-actin is shown as a loading control. Each lane represents a different mTAL cell culture. Densitometric analysis (relative to WT) is shown next to the blots. Bars indicate mean ± s.e.m.; **p = 0.01 (Unpaired two-tailed t test). (f) Representative immunofluorescence analysis for phosphorylated NKCC2 (at position Thr96, red) in permeabilized mTAL cells derived from Hlb320 and WT mice. Z-plan reconstructions are shown on the picture borders. Nuclei are stained in blue with DAPI. Scale bar: 25 µm. (g) Transepithelial voltage (Vte, mV) recordings using a chopstick electrode on mTAL cells derived from Hlb320 (red), WT (black) mice and WT cultures after application of apical bumetanide (100 µM; white). Each dot represents a filter of primary TAL cells. Bars indicate mean ± s.e.m.; ****p = 0.0001 (Unpaired two-tailed t test). (h) Evolution of transepithelial voltage (Vte, mV) after basolateral ddAVP application (0.1 µM) in mTAL cells derived from Hlb320 (red) or WT (black) mice. Each dot represents a mTAL cell culture. Bars indicate mean ± s.e.m.; *p = 0.05; **p = 0.01 (Unpaired two-tailed t test). The RT-PCR amplification product for Avpr2 is shown for Hlb320 and WT mTAL cultures. Gapdh is shown as a loading control. Uncropped image of blots can be found in Supplementary Figure S8.

Fig 4: Bartter-like phenotype in NaCl loaded Hlb320 mice. (a) Evolution of physiological parameters related to kidney function in Hlb320 (red) and WT (black) mice in control conditions and after NaCl loading, as indicated. Each dot represents a mouse. Bars indicate mean ± s.e.m.; *p = 0.05; **p = 0.01; ***p = 0.001; ****p = 0.0001 (Unpaired two-tailed t test). (b) Left panel: Systolic blood pressure (SBP) averages as assessed by tail cuff measurements on the same mice during 2 months. Each single dot represents an average of 3–5 consecutive daily SBP recordings in Hlb320 (red) and WT (black) mice at control conditions and at different reporting periods after NaCl loading, as indicated. Bars indicate mean ± s.e.m.; ***p = 0.001 (Unpaired two-tailed t test); ††p = 0.01 (Paired two-tailed t test: SBP at 2 months of NaCl loading versus baseline). Right panel: Pressure-natriuresis relationship obtained by plotting Na+ excretion (µmol/24 h) against SBP for Hlb320 (red) and WT (black) mice at control conditions and after NaCl loading, as indicated. Error bars indicate means ± s.d.

Fig 5: Uromodulin accumulation in vivo and consequences in the kidney. (a) Western blot analysis of total kidney uromodulin from Hlb320 and WT mice. ß-actin is blotted as a loading control and densitometric analysis is shown next to the blot. Bars indicate mean ± s.e.m.; ***p = 0.001 (Unpaired two-tailed t test).(b) Upper panels: Western blot analysis for uromodulin on kidney fractions enriched in large membranes/plasma membrane (“membrane fraction”) from Hlb320 and WT mice. Integral plasma membrane protein flotillin-1 and GAPDH are blotted as positive and negative controls, respectively. Densitometric analysis with normalization to flotillin-1 is shown next to the blot. Bars indicate mean ± s.e.m.; *p = 0.05 (Unpaired two-tailed t test). Lower panels: Western blot analysis for uromodulin on kidney fractions enriched in cytoplasmic proteins/small vesicles (“cytosolic fraction”) from Hlb320 and WT mice. Cytosolic protein GAPDH and flotillin-1 are blotted as positive and negative controls, respectively. Densitometric analysis with normalization to GAPDH is shown next to the blot. Bars indicate mean ± s.e.m.; **p = 0.01 (Unpaired two-tailed t test). (c) Representative immunofluorescence analysis for uromodulin (green) and ER chaperone calnexin (red) and Golgi protein GM130 (red) on kidney sections from Hlb320 and WT mice, as indicated. Nuclei are stained in blue with DAPI. Scale bar: 50 µm and 25 µm for co-localization studies; G, glomerulus. (d) Transcript levels of major renal transporters and channels as assessed by RT-qPCR on total kidney extracts from Hlb320 mice and WT mice. A normalization factor based on 6 different housekeeping genes was used and results are expressed relative to WT expression (red dotted line). N = 4. Bars indicate mean ± s.e.m. Lrp2, megalin; Aqp1, aquaporin-1; Slc12a1a, NKCC2A; Slc12a1b, NKCC2B; Slc12a1f, NKCC2F; Kcnj1, ROMK; Umod, uromodulin; Slc12a3, NCC; Aqp2, aquaporin-2; Scnn1b, ß-ENaC; Scnn1c, ?-ENaC. (e) Western blot analysis in total kidney lysates from Hlb320 and WT mice. A representative ß-actin blot is shown as loading control. Densitometric analysis (relative to WT kidneys: red dotted line) is shown below the blots. Bars indicate mean ± s.e.m.; *p = 0.05; ***p = 0.001 (Unpaired two-tailed t test). (f) Representative immunofluorescence analysis for uromodulin (green) and phosphorylated NKCC2 (at position Thr96, red) in kidney sections from Hlb320 and WT mice. Nuclei are stained in blue with DAPI. Scale bar: 50 µm; G, glomerulus. (g) Western blot analysis for phosphorylated SPAK (Ser373)/phosphorylated OSR1 (Ser325) and total SPAK in total kidney lysates from Hlb320 and WT mice. ß-actin is blotted as a loading control. Densitometric analysis reports levels normalized to ß-actin in Hlb320 kidneys relative to WT kidneys. Bars indicate average ± s.e.m.; **p = 0.01, ****p = 0.0001 (Unpaired two-tailed t test). Uncropped image of blots can be found in Supplementary Figure S8.