The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin
- San Raffaele Scientific Institute, Italy
- Karolinska Institutet, Sweden
- FIRC Institute of Molecular Oncology, Italy
- University of Zurich, Switzerland
- Lerner Research Institute, United States
- University of Lausanne, Switzerland
Figures
Figure 1
MDCK cells as a model to study physiological uromodulin shedding.
(A) Schematic representation of human uromodulin domain structure containing a leader peptide (predicted to be cleaved at residue 23), three EGF-like domains, a central domain with 8 conserved …
Figure 2 with 2 supplements
A serine protease is responsible for the release of polymerisation-competent uromodulin.
(A) Immunofluorescence analysis showing uromodulin on the surface of MDCK cells treated with vehicle (DMSO) (ctr), protease inhibitor cocktail (PIC) or single PIC components, as indicated. Scale …
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Figure 2—source data 1
Quantification of the area of uromodulin polymers on the surface of MDCK cells after protease inhibitor treatment (Figure 2A).
- https://doi.org/10.7554/eLife.08887.005
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Figure 2—source data 2
Short cleavage inhibition by HAI-1 expression (Figure 2—figure supplement 2C).
- https://doi.org/10.7554/eLife.08887.006
Figure 2—figure supplement 1
PIC treatment does not affect uromodulin intracellular distribution and expression.
(A) Immunofluorescence analysis showing intracellular distribution of uromodulin in MDCK cells treated with vehicle (DMSO) (ctr) or protease inhibitor cocktail (PIC). KDEL is a marker of the …
Figure 2—figure supplement 2
Expression of the serine protease inhibitor HAI-1 effectively reduces uromodulin cleavage at the urinary site.
(A) Immunofluorescence analysis showing uromodulin on the surface of MDCK cells co-expressing the serine protease inhibitor HAI-1 (Hepatocyte growth factor Activator Inhibitor-1), as indicated. …
Figure 3 with 1 supplement
Hepsin and prostasin interact with uromodulin to induce its cleavage and polymerisation.
(A) Immunofluorescence analysis (extended focus image) showing uromodulin on the surface of HEK293 cells. The protein is not assembled into polymers when expressed in this cellular system. Scale …
Figure 3—figure supplement 1
Hepsin and prostasin expression in MDCK and HEK293 cells.
(A) RT-PCR analysis showing gene expression of candidate proteases hepsin (HPN) and prostasin (PRSS8) in MDCK and HEK293 cells. Constructs containing coding sequences of the human proteases were …
Figure 4
Hepsin and prostasin directly cleave uromodulin in vitro.
(A) Schematic representation of human uromodulin domain structure as shown in Figure 1A. The region not included in recombinant efUmod is shadowed. (B) The deletion of the elastase-sensitive …
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 …
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Figure 5—source data 1
Transcript level of HPN and PRSS8 in MDCK cells after shRNA transfection (Figure 5B).
- https://doi.org/10.7554/eLife.08887.013
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Figure 5—source data 2
Quantification of the area of uromodulin polymers on the surface of MDCK cells after shRNA transfection (Figure 5C).
- https://doi.org/10.7554/eLife.08887.014
Figure 6
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 …
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Figure 6—source data 1
Transcript level of Hpn in mouse microdissected nephron segments (Figure 6A).
- https://doi.org/10.7554/eLife.08887.016
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Figure 6—source data 2
Quantification of urinary uromodulin secretion in Hpn-/- and control mice (Figure 6C).
- https://doi.org/10.7554/eLife.08887.017
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Figure 6—source data 3
Quantification of uromodulin levels in kidney lysates of Hpn-/- and control mice (Figure 6D).
- https://doi.org/10.7554/eLife.08887.018
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Figure 6—source data 4
Transcript levels of Umod in Hpn-/- and control mice (Figure 6F).
- https://doi.org/10.7554/eLife.08887.019
Figure 7 with 2 supplements
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 …
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Figure 7—source data 1
Transcript level of Prss8 in mouse microdissected nephron segments (Figure 7—figure supplement 2A).
- https://doi.org/10.7554/eLife.08887.021
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Figure 7—source data 2
Quantification of urinary uromodulin secretion in Prss8-/- and control mice (Figure 7—figure supplement 2C).
- https://doi.org/10.7554/eLife.08887.022
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 …
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 …
Figure 8
Model of uromodulin shedding and polymerisation.
Uromodulin is exclusively expressed by TAL tubular epithelial cells. The protein enters the secretory pathway and reaches the plasma membrane in a polymerisation-incompetent conformation. This is …
Author response image 1
Mass spectrometry analysis of the long uromodulin isoform released by MDCK cells.
(A) Mass spectrometry sequence coverage (42%) of Asp-N-digested human uromodulin (long isoform) from the medium of stably expressing MDCK cells. The analysis was carried out as described in the …
Author response image 2
Expression level of renal genes in Hpn-/- mice.
Quantification by Real-time qPCR of the mRNA levels of cyclooxygenase 2 (Cox2) (Ptgs2), sodium-chloride co-transporter (NCC) (Slc12a3) and the renal outer medullary potassium channel (ROMK) (Kcjn1) …
Author response image 3
Yeast agglutination assays on uropathogentic E. coli clinical isolates.
Representative images of yeast agglutination assay. The assay was performed incubating 10 µl of previously identified bacteria isolates (haemagglutination-positive isolates #9, 12, 10), grown in …
Tables
Table 1
Primers (5'-3') used to generate UMOD and protease constructs.
| efUMOD-PCR1 T7: TAATACGACTCACTATAGGG Reverse: ACACGTCCCCTCCACGTGGTGATGGTGATGATGAC |
| efUMOD-PCR2 Forward: CATCACCATCACCACGTGGAGGGGACGTGTGAGGA BGHrev: TAGAAGGCACAGTCGAGG |
| efUMOD_sol Forward: GGACAGATCTACGTCGACGGGACGTGTGAGGAGTGCAG Reverse: CCGGAATTCCTAGTGATGATGGTGATGATGCTGGACACCTTTCCGTGTG |
| efUMOD_AYAA Forward: CCTACCTGCTCTGGGACCGCATACGCAGCTGGGAGTGTCATAGATCAATCCC Reverse: GGGATTGATCTATGACACTCCCAGCTGCGTATGCGGTCCCAGAGCAGGTAGG |
| efUMOD_N513Q Forward: CACCCAGTAGCCAGGCCACGGACCCCC Reverse: GGGGGTCCGTGGCCTGGCTACTGGGTG |
| PRSS8_S238A Forward: GCCAGGGTGACGCTGGGGGCCCA Reverse: TGGGCCCCCAGCGTCACCCTGGC |
| HPN_S353A Forward: CTGCCAGGGCGACGCCGGTGGTCCCTTT Reverse: AAAGGGACCACCGGCGTCGCCCTGGCAG |
Table 2
Oligonucleotide sequences used to generate shRNAs.
| Target gene | Target position from ATG codon | siRNA oligo (5’-3’) |
|---|---|---|
| HPN_siRNA#1 | 122-140 | CCTTCCTACTCAAGAGTGA |
| HPN_siRNA#2 | 1195-1213 | TGGATCTTCCAGGCCATAA |
| PRSS8_siRNA#1 | 258-276 | GAAGGAAGACTATGAGGTA |
| PRSS8_siRNA#2 | 593-611 | TGTACAACATCAACGCTAA |
| Control siRNA | NA | TAGTGAGATTCGTTAAGAT |
Table 3
Primers used for gene expression analysis (RT-PCR).
| Gene | PCR product length | Primer (5’-3’) |
|---|---|---|
| HPN (Dog / Human) | 353 bp | Forward: GCTGCGAGGAGATGGGCTTC Reverse: CGGGAAGCAGTGGGCGGCTG |
| PRSS8 (Dog / Human) | 547 bp | Forward: CCTGGCAGGTCAGCATCACC Reverse: CCAGAGTCACCCTGGCAGGC |
| GAPDH (Human) | 314 bp | Forward: CCACCCAGAAGACTGTGGAT Reverse: GTTGAAGTCAGAGGAGACCACC |
| GAPDH (Dog) | 289 bp | Forward: CTCTGGGAAGATGTGGCGTGAC Reverse: GTTGAAGTCACAGGAGACCACC |
Table 4
Primers used for gene expression analysis (Real-Time qPCR).
| Gene | PCR product length | Primer (5’-3’) |
|---|---|---|
| HPN (Dog) | 164 bp | Forward: TGGTCCACCTGTCCAGCCCC Reverse: GACTCGGGCCTCCTGGAGCA |
| Hpn (Mouse) | 152 bp | Forward: CTGACTGCTGCACATTGCTT Reverse: GGGTCTCGAAAGGGAAGGTA |
| PRSS8 (Dog) | 156 bp | Forward: TCCGGACTTGCCTTCTGCGGT Reverse: AGCTGAGAGCACCCACTGCTCA |
| GAPDH (Dog) | 289 bp | Forward: CTCTGGGAAGATGTGGCGTGAC Reverse: GTTGAAGTCACAGGAGACCACC |
| Prss8 (Mouse) | 147bp | Forward: ATCACCCACTCAAGCTACCG Reverse: AGTACAGTGAAGGCCGTTGG |
| Umod (Mouse) | 207 bp | Forward: ATGGACCAGTCCTGTCCTG Reverse: CCGTCTCGCTTCTGTTGAGT |
| Hprt1 (Mouse) | 162 bp | Forward: ACATTGTGGCCCTCTGTGTG Reverse: TTATGTCCCCCGTTGACTGA |
