Figure 7. Absence of hepsin in vivo abolishes physiological cleavage and polymerisation of uromodulin.

(A) Representative Western blot analysis of N-deglycosylated urinary uromodulin secreted by Hpn^-/- mice or control animals. Hpn^-/- mice show the presence of two uromodulin isoforms: a short one with similar electrophoretic mobility as in wild-type urines (white arrowhead), and a longer one that is absent in samples from wild-type mice (black arrowhead) (n = 6/group). (B) Mass spectrometry (MS) sequence coverage (52% over the entire protein) of trypsin-digested mouse uromodulin (short isoform) (UniProt accession Q91X17) purified from urine of Hpn^-/- mice. Matching peptides are shown in red, while the C-terminal peptide is shown in blue. This peptide ends at R607, a distal C-terminal residue with respect to the one reported for mouse urinary uromodulin (F588 [Santambrogio et al., 2008]). (C) Representative tandem mass-spectrometry (MS/MS) spectrum, confirming the identity of the identified C-terminal peptide (⁵⁹⁹VLNLGPITR⁶⁰⁷) of the short uromodulin isoform released by Hpn^-/- mice, and table of fragmented ions. (D) Schematic representation of mouse uromodulin domain structure as in Figure 1A. The blow-up shows that in the absence of hepsin, the cleavage generating the short uromodulin isoform is abolished and alternative ones at more C-terminal sites (distal to R607) take place. (E) Representative Western blot analysis of uromodulin in supernatant (SN) and pellet (P) fractions from a polymerisation assay performed on urinary samples from Hpn^-/- mice or control animals. Urinary uromodulin from control animals is precipitated in the pellet fraction, reflecting full engagement in polymeric structures, while the one from Hpn^-/- mice is only detected in the supernatant (n = 4/group).

DOI: http://dx.doi.org/10.7554/eLife.08887.020

Figure 7—figure supplement 1. Urinary uromodulin misprocessing in Hpn^-/- mice.

(A) Mass spectrometry (MS) sequence coverage (51% over the entire protein) of trypsin-digested mouse uromodulin (long isoform) (UniProt accession Q91X17) purified from urine of Hpn^-/- mice. Matching peptides are shown in red, while the C-terminal peptide is shown in blue. This peptide ends at K616, a distal C-terminal residue with respect to the one reported for mouse urinary uromodulin (F588 [Santambrogio et al., 2008]). (B) Representative tandem mass-spectrometry (MS/MS) spectrum, confirming the identity of the identified C-terminal peptide (⁶⁰⁸QGVQASVSK⁶¹⁶) of the long uromodulin isoform released by Hpn^-/- mice, and table of fragmented ions.

DOI: http://dx.doi.org/10.7554/eLife.08887.023

Figure 7—figure supplement 2. Uromodulin secretion is not affected by lack of prostasin in vivo.

(A) Transcript level of Prss8, as assessed by Real-Time qPCR on microdissected nephron segments (normalised to Gapdh). Expression of Prss8 is detected in proximal convoluted tubules (PCT), proximal straight tubules (PST) and, to a lesser extent, in thick ascending limb (TAL) and collecting ducts (CD). Bars indicate average ± s.e.m. of 3 independent experiments (Figure 7—source data 1). (B) Immunofluorescence analysis of mouse kidney sections shows strong signal of endogenous prostasin on the apical plasma membrane of proximal tubules, and weak signal on the apical plasma membrane of TAL epithelial cells where it co-localises with uromodulin. Scale bar, 20 µm. (C) Representative Western blot analysis of urinary uromodulin from control Prss8^lox/loxor Prss8^-/- mice. Urinary protein loading was normalised to urinary creatinine concentration. Densitometric analysis shows that uromodulin secretion is comparable between Prss8^-/- mice and control Prss8^lox/loxanimals (average ± s.d., n = 5/group, Figure 7—source data 2) (Student’s t test). (D) Representative Western blot analysis of N-deglycosylated urinary uromodulin secreted by Prss8^-/- mice or control animals. An isoform of identical molecular weight, corresponding to the short uromodulin isoform, is detected in urine samples of both genotypes (n = 5/group). (E) Mass spectrometry sequence coverage (55% over the entire protein) of AspN-digested mouse uromodulin (UniProt accession Q91X17) purified from urine of Prss8^-/- mice. Matching peptides are shown in red, while the C-terminal peptide is shown in blue. This peptide ends at F588, the same C-terminal residue identified in urinary uromodulin of wild-type mice (Santambrogio et al., 2008) and control Prss8^lox/loxanimals (data not shown). (F) Representative MS/MS spectrum confirming the sequence of urinary uromodulin C-terminal peptide (⁵⁷³DSTSEQCKPTCSGTRF⁵⁸⁸) in Prss8^-/- mice and table of fragmented ions.

DOI: http://dx.doi.org/10.7554/eLife.08887.024