Quantitative Proteomic Study Unmasks Fibrinogen Pathway in Polycystic Liver Disease Ⅲ

4. Discussion 

Polycystic liver disease (PLD) is commonly an extrarenal manifestation associated with autosomal dominant polycystic kidney disease (ADPKD). PLD is characterized by progressive dilation of the bile ducts and progression of multiple cysts, occupying at least half the volume of the liver parenchyma. Several of the current therapeutic strategies are based on surgical and pharmacological procedures to improve the symptoms of the disease. For this reason, the treatment of PLD has been ineffective so far, and the only curative option is liver transplantation [45]. This is mainly because the key intrinsic molecular mechanism of cystogenesis remains unknown. The main approaches are focused on the cAMP signaling pathway and somatostatin analogs, and several new targets (for example, histone deacetylase 6, Cdc25A phosphatase, PPAR-γ, and matrix metalloproteases) have been evaluated in preclinical studies, but need to be tested clinically [8]. So, an emerging interest in understanding molecular pathways and developing new therapeutic strategies is increasing [2,8]. The purpose of the present and novel SWATH-MS work was characterized by the proteomic changes in polycystic livers and to provide new insights and therapeutic targets for PLD.

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In previous studies, we have identified in an orthologous model of ADPKD (Pkd1cond/cond; Tam-Cre) a developmental window for kidney cystogenesis, suggesting that the timing of secondary events may influence the severity of cystic kidney disease. Using the same strategy, we investigated whether this window of renal development corresponded in time with a similar window of hepatic development. Interestingly, we observed that cystic liver phenotype does not follow the same timing, suggesting that the kidney and liver have possibly different mechanisms undergoing cystogenesis. Further studies should be focused on identifying the common and different mechanisms of disease in both organs and identifying the unknown liver developmental window for cystogenesis. For further analysis, we used a next-generation quantitative proteomic approach SWATH-MS to perform differential proteome analysis of cystic liver disease in a mild and more severe PLD phenotype within the same kidney developmental window. Our data suggest that in advanced stages of disease progression, the number of pathways deregulated are increased, probably due to secondary effects of the disease. In the same way, these results reinforce the idea that cyst growth passes with two big stages: cyst initiation and cyst progression [46]. Interestingly, both proteome analyses from a mild (p14 group) and a severe (p12 group) cystic status identified protein complexes of fibrinolysis directly related to liver cystic disease. At the milder stages of the disease, we identified ANXA2 as one of the interesting proteins to validate as a potential therapeutic target for PLD. ANXA2 is a calcium-regulated membrane-binding protein produced by a wide range of cell types, including epithelial, dendritic, trophoblast, and tumor cells, as well as monocytes and macrophages. The main role is to maintain cell surface proteolytic activity, but also fulfills a range of intracellular functions, including exocytosis, endocytosis, membrane repair [47] and maintenance of adherent-like intercellular junctions [48]. Its numerous functions contribute to fibrinolysis, regulation of inflammation and immune system activation, and tissue injury and repair; as a result, ANXA2 dysfunction has been implicated in multiple human diseases, for example, extracellular matrix remodeling and hepatic fibrosis [49,50], which is a common feature in disease progression of PLD [8]. Interestingly, at the severe stage of liver disease, our proteome analysis discovered a number of different proteins related to the fibrinogen complex. During tissue and vascular injury, fibrinogen is converted enzymatically by thrombin to fibrin, and during fibrinolysis, fibrin is degraded by the main enzyme plasmin (the active form of plasminogen). Fibrinogens are associated with MAPK [51,52] and integrins [53], two pathways that have been shown to be effective as potential therapy for ADPKD in preclinical studies [54,55]. Even the animal model of one of the overexpressed fibrinogens (FIBA) has been shown to have cystic disease [56]. Moreover, the increased activity of fibrinogen clusters in PLD offers a new perspective to treat the disease in comparison to the current experimental drugs [57]. Consequently, the decrease in this cluster could support a new mechanism of action for drugs in which fibrinogen family proteins would be the therapeutic targets. A greater example would be the blockade of ERRγ (estrogen-related receptor γ), an orphan nuclear receptor [58,59], which modulates fibrinogen levels in hypofibrinogenemia states caused by diet-induced obesity, diabetes mellitus type 2, liver injury, and alcohol-induced oxidative stress [60,61]. Specifically, inverse agonists, such as GSK5182 (CAS: 877387-37-6) [60–66] and DN200434 [67,68], would offer such beneficial action through the decrease of fibrinogen levels. 

In summary, we identified several novel targets and pathways involved in the physiopathology of PLD by quantitative proteomic SWATH-MS technology. We emphasized the novel therapeutic opportunity presented by fibrinogens and fibrinolysis (and the other related identified proteins), mainly due the fact that all validations with significant differences carried out are related to the fibrinogen complex. Nevertheless, there is potential to confirm this in future preclinical studies as well as more functional studies to characterize the exact role of our target candidates in PLD. In conclusion, this work has created a new mechanism and opportunity for future research in PLD physiopathology, leading to possible new therapeutic approaches of the disease. 

5. Summary 

We reported a novel proteomic quantitative study based on SWATH-MS technology comparing proteomes of wild-type and polycystic livers. Several novel pathways and targets were identified, expanding the knowledge about molecular mechanisms in the PLD field and highlighting the fibrinogen complex as a possible new therapeutic target.

Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biomedicines10020290/s1, Figure S1: Qualitative DDA mass spectrometry analysis in the different groups of the study, Figure S2: TAS (Total Area Sums) normalization plots p14 group, Figure S3: TAS (Total Area Sums) normalization plots of p12 group, Figure S4: Heatmap figure of unsupervised cluster analysis of proteins with significant differences according to SWATH-MS analysis, Figure S5: Unsupervised heatmap cluster analysis of all proteins detected by SWATH-MS, Figure S6: Unsupervised PCAs from SWATH-MS data, Figure S7: GO enrichment and pathway analysis of protein significantly regulated in hepatic cystogenesis according to String and Reactome, respectively, Figure S8: Full protein-protein interaction map according String, Figure S9: Box-plots of quantitative proteomic SWATH-MS data of selected targets for validation, Table S1: ID Analyzed samples, distribution in each group and samples pooling for the acquisition of shotgun runs to build the spectral library, Table S2: Primers sequence list used for RT-qPCR analysis, Table

Author Contributions: Conceptualization, M.A.G.-G. and A.C.; methodology, A.C., M.V.-G., L.N.-G., A.M.-V., M.d.P.C.-V. and S.B.B.; formal analysis, A.C.; resources: M.A.G.-G. and S.B.B.; data curation, A.C.; writing—original draft preparation, A.C.; writing—review and editing, M.A.G.-G., S.B.B. and L.N.-G.; visualization, M.A.G.-G. and A.C.; supervision, M.A.G.-G.; project administration, M.A.G.- G.; funding acquisition, M.A.G.-G. All authors have read and agreed to the published version of the manuscript. 

Funding: This research was funded by Instituto de Salud Carlos III under FIS/FEDER funds PI15/01467 and PI18/00378 (to M.A.G.-G.) and by Xunta de Galicia award IN607B 2016/020 (to M.A.G.-G.). Salary support was provided by a Xunta de Galicia predoctoral fellowship 2016 to A.C. and a Xunta de Galicia predoctoral fellowship 2017 to M.V.-G. 

References 

1. Cnossen, W.R.; Drenth, J.P.H. Polycystic liver disease: An overview of pathogenesis, clinical manifestations and management. Orphanet J. Rare Dis. 2014, 9, 1–13. [CrossRef] [PubMed] 

2. Santos-Laso, A.; Izquierdo-Sánchez, L.; Lee-Law, P.Y.; Perugorria, M.J.; Marzioni, M.; Marin, J.J.G.; Bujanda, L.; Banales, J.M. New Advances in Polycystic Liver Diseases. Semin. Liver Dis. 2017, 37, 45–55. [CrossRef] [PubMed] 

3. Suwabe, T.; Shukoor, S.; Chamberlain, A.M.; Killian, J.M.; King, B.F.; Edwards, M.; Senum, S.R.; Madsen, C.D.; Chebib, F.T.; Hogan, M.C.; et al. Epidemiology of autosomal dominant polycystic kidney disease in Olmsted county. Clin. J. Am. Soc. Nephrol. 2020, 15, 69–79. [CrossRef] [PubMed]

4. Cordido, A.; Besada-Cerecedo, L.; García-González, M.A. The Genetic and Cellular Basis of Autosomal Dominant Polycystic Kidney Disease—A Primer for Clinicians. Front. Pediatr. 2017, 5, 279. [CrossRef] [PubMed]

5. Bergmann, C.; Guay-Woodford, L.M.; Harris, P.C.; Horie, S.; Peters, D.J.M.; Torres, V.E. Polycystic kidney disease. Nat. Rev. Dis. Prim. 2018, 4, 1–24. [CrossRef]

6. Cordido, A.; Vizoso-Gonzalez, M.; Garcia-Gonzalez, M.A. Molecular Pathophysiology of Autosomal Recessive Polycystic Kidney Disease. Int. J. Mol. Sci. 2021, 22, 6523. [CrossRef]

7. Van Aerts, R.M.M.; van de Laarschot, L.F.M.; Banales, J.M.; Drenth, J.P.H. Clinical management of polycystic liver disease. J. Hepatol. 2018, 68, 827–837. [CrossRef]

8. Perugorria, M.J.; Masyuk, T.V.; Marin, J.J.; Marzioni, M.; Bujanda, L.; Larusso, N.F.; Banales, J.M. Polycystic liver diseases: Advanced insights into the molecular mechanisms. Nat. Rev. Gastroenterol. Hepatol. 2014, 11, 750–761. [CrossRef] 

9. Banales, J.M.; Masyuk, T.V.; Gradilone, S.A.; Masyuk, A.I.; Medina, J.F.; LaRusso, N.F. The cAMP effectors Epac and protein kinase a (PKA) are involved in the hepatic cystogenesis of an animal model of autosomal recessive polycystic kidney disease (ARPKD). Hepatology 2009, 49, 160–174. [CrossRef]

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