Opposing Expressions of Nidogen-1 and Cdc42 Genes in the Development of Liver Cirrhosis in Patients with Non-Alcoholic Fatty Liver Disease

² Student Research Committee, Hamadan University of Medical Sciences, Hamadan, Iran

³ Department of Clinical Biochemistry, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran

*Corresponding author: Sina Mohagheghi, Department of Clinical Biochemistry, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran. Email: amr.mohaghegh@yahoo.com

EXTENDED ABSTRACT

Background

Cirrhosis is the advanced consequence of persistent hepatic injury, in which normal liver parenchyma is progressively replaced by fibrotic tissue [1]. Chronic viral hepatitis, non-alcoholic fatty liver disease (NAFLD), autoimmune hepatitis (AIH), cholestatic disorders, and primary sclerosing cholangitis (PSC) are among the causes described in the source article [2–6]. NAFLD spans simple steatosis, non-alcoholic steatohepatitis (NASH), fibrosis, and ultimately cirrhosis; the article notes that approximately 10–25% of patients with NASH may develop cirrhosis [7]. Because cirrhosis may remain clinically silent until liver injury becomes advanced and because liver biopsy, although important for staging and uncertain diagnoses, is invasive, biological pathways involved in fibrosis remain relevant targets for investigation [8–11, 24]. The extracellular matrix includes collagen, elastin, fibronectin, proteoglycans, glycosaminoglycans, laminin, and other glycoproteins. Nidogen-1 constitutes approximately 2–3% of basement-membrane proteins and links laminin and collagen networks, contributing to basement-membrane formation and stability [12–14]. Experimental studies cited by the article suggest protective and anti-fibrotic effects of Nidogen-1 in ischemic and injured tissues [15, 16]. Cdc42, a Rho-family GTPase, regulates transcription, the cell cycle, cytoskeletal organization, and survival, but published data have suggested both pro-fibrotic and anti-fibrotic actions depending on the tissue and experimental context [17–19]. Accordingly, this study examined hepatic Nidogen-1 and Cdc42 gene expression in cirrhosis of different etiologies, simple non-alcoholic steatosis, and control liver tissue.

Methods

This case-control study analyzed 38 liver tissue samples. The cirrhosis group comprised 24 adults who underwent liver transplantation at the Namazi Hospital Transplant Center of Shiraz University of Medical Sciences. Cirrhosis was divided equally into four etiologic subgroups: NASH-related cirrhosis (n=6), viral hepatitis-related cirrhosis caused by HBV or HCV (n=6), AIH-related cirrhosis (n=6), and PSC-related cirrhosis (n=6). Eligibility for the cirrhosis group included age >18 years, one of the specified causes of cirrhosis, and absence of other causes of cirrhosis. The second group contained six liver tissues with simple steatosis. The control group contained eight normal liver samples obtained from adults undergoing hepatectomy; control participants had no history of chemotherapy or other treatments affecting benign tumors. After surgical removal, liver tissue intended for molecular analysis was washed in normal saline, frozen in liquid nitrogen, and stored at -80 °C. A separate portion was fixed in 4% formalin and embedded in paraffin. Paraffin-fixed liver samples were sectioned at 4–5 µm, stained with hematoxylin and eosin, and evaluated by a liver pathologist, who diagnosed cirrhosis and simple steatosis and confirmed normal histology in controls. Simple steatosis was defined as >5% steatosis without inflammation or fibrosis. Total RNA was extracted using the RNeasy kit (catalogue no. 74104; Qiagen, Germany), reverse-transcribed to cDNA with a Thermo Fisher Scientific cDNA synthesis kit, and analyzed by RT-qPCR using 1 µL cDNA on a LightCycler 96 Real-Time PCR System (Roche, Germany). Beta-actin served as the internal reference gene, and relative expression was calculated using the 2^-ΔCT method. Statistical analysis was performed in SPSS version 16. Normality was assessed using the Shapiro-Wilk test; independent t tests and one-way ANOVA were used for normally distributed comparisons, whereas Kruskal-Wallis and Mann-Whitney U tests were applied to non-normal data. Results were reported as mean ± SEM, and P<0.05 was considered statistically significant. GraphPad Prism version 10 was used for graphical presentation. The study was approved by the Ethics Committee of Hamadan University of Medical Sciences (IR.UMSHA.REC.1403.248); procedures followed the 1964 Declaration of Helsinki, and informed consent was obtained from all participants.

Results

The six study subgroups comprised eight controls, six patients with simple steatosis, and four cirrhosis subgroups of six participants each. Male participants numbered 3 in the control group, 3 in simple steatosis, 4 in NASH cirrhosis, 1 in PSC cirrhosis, 2 in AIH cirrhosis, and 5 in HBV/HCV cirrhosis. The demographic values reported in the original Table 2 are reproduced below without renumbering.

Table 2. Demographic characteristics of patients with cirrhosis, simple steatosis, and the control group.

Across all cirrhotic liver tissues, Nidogen-1 and Cdc42 expression were both significantly lower than in control liver tissue (P=0.001 and P=0.008, respectively). When cirrhosis was analyzed by etiology, Nidogen-1 expression was lower in every cirrhotic subgroup than in controls, but the decrease reached statistical significance only for PSC-related cirrhosis and AIH-related cirrhosis (P=0.003 for each comparison). Among the cirrhosis subgroups, the highest Nidogen-1 expression was observed in NASH-related cirrhosis and the lowest in PSC-related cirrhosis. Cdc42 expression likewise decreased in all cirrhosis subgroups relative to controls; this reduction was statistically significant for AIH-related cirrhosis (P=0.01) and viral hepatitis-related cirrhosis (P=0.018). Among cirrhotic etiologies, Cdc42 expression was highest in NASH-related cirrhosis and lowest in AIH-related cirrhosis. The comparison focused on NAFLD progression showed a different pattern for the two genes. Nidogen-1 expression did not differ significantly among controls, simple steatosis, and NASH-related cirrhosis; nevertheless, the reported trend was highest expression in controls, an intermediate level in simple steatosis, and the lowest level in NASH cirrhosis. By contrast, Cdc42 expression differed significantly among these three groups (P=0.001). Cdc42 expression in simple steatosis was significantly lower than in controls (P<0.001) and was also significantly lower than in NASH-related cirrhosis (P=0.043). Thus, while both genes showed overall downregulation in cirrhosis compared with healthy liver tissue, their expression trajectories between simple steatosis and NASH cirrhosis were not parallel.

Conclusion

The study demonstrates reduced hepatic expression of both Nidogen-1 and Cdc42 in cirrhosis, with etiologic differences and opposing patterns during the transition from simple steatosis to NASH cirrhosis. In the context of the experimental evidence cited by the authors, the progressive decrease in Nidogen-1 from control tissue through simple steatosis to NASH cirrhosis is compatible with a predominantly anti-fibrotic role [15, 16, 25, 26]. For Cdc42, the increase from simple steatosis to NASH cirrhosis, despite lower overall expression in cirrhotic tissue than in controls, was interpreted as consistent with possible participation in fibrosis formation; prior studies cited in the article have reported both fibrogenic and inhibitory effects of Cdc42 in other tissues [19, 27–29]. These interpretations remain limited by the small sample size and the absence of direct measurement of Nidogen-1 and Cdc42 protein levels.

Keywords: Cdc42, Cirrhosis, Nidogen, Simple Steatosis

REFERENCES

1. Ginès P, Krag A, Abraldes JG, Solà E, Fabrellas N, Kamath PS. Liver cirrhosis. Lancet. 2021;398(10308):1359-1376. PMID: 34543610 DOI: 10.1016/S0140-6736(21)01374-X
2. Lădaru A, Bălănescu P, Stan M, Codreanu I, Anca IA. Candidate proteomic biomarkers for non-alcoholic fatty liver disease (steatosis and non-alcoholic steatohepatitis) discovered with mass-spectrometry: a systematic review. Biomarkers. 2016;21(2):102-14. PMID: 26632636 DOI: 10.3109/1354750X.2015.1118542
3. Wong VW, Wong GL, Choi PC, Chan AW, Li MK, Chan HY, et al. Disease progression of non-alcoholic fatty liver disease: a prospective study with paired liver biopsies at 3 years. Gut. 2010;59(7):969-74. PMID: 20581244 DOI: 10.1136/gut.2009.205088
4. Webb GJ, Hirschfield GM, Krawitt EL, Gershwin ME. Cellular and Molecular Mechanisms of Autoimmune Hepatitis. Annu Rev Pathol. 2018;13:247-92. PMID: 29140756 DOI: 10.1146/annurev-pathol-020117-043534
5. Karlsen TH, Folseraas T, Thorburn D, Vesterhus M. Primary sclerosing cholangitis-a comprehensive review. J Hepatol. 2017;67(6):1298-323. PMID: 28802875 DOI: 10.1016/j.jhep.2017.07.022
6. Tabibian JH, Bowlus CL. Primary sclerosing cholangitis: A review and update. Liver Res. 2017;1(4):221-230. PMID: 29977644 DOI: 10.1016/j.livres.2017.12.002
7. Brunt EM, Neuschwander-Tetri BA, Oliver D, Wehmeier KR, Bacon BR. Nonalcoholic steatohepatitis: histologic features and clinical correlations with 30 blinded biopsy specimens. Hum Pathol. 2004;35(9):1070-82. PMID: 15343508 DOI: 10.1016/j.humpath.2004.04.017
8. Parola M, Pinzani M. Liver fibrosis: Pathophysiology, pathogenetic targets and clinical issues. Mol Aspects Med. 2019;65:37-55. PMID: 30213667 DOI: 10.1016/j.mam.2018.09.002
9. Aubé C, Oberti F, Korali N, Namour MA, Loisel D, Tanguy JY, et al. Ultrasonographic diagnosis of hepatic fibrosis or cirrhosis. J Hepatol. 1999;30(3):472-8. PMID: 10190731 DOI: 10.1016/S0168-8278(99)80107-X
10. Heidelbaugh JJ, Bruderly M. Cirrhosis and chronic liver failure: part I. Diagnosis and evaluation. Am Fam Physician. 2006;74(5):756-62. PMID: 16970019
11. Elias H, Bengelsdorf H. The structure of the liver of vertebrates. Acta Anat (Basel). 1952;14(4):297-337. PMID: 14943381 DOI: 10.1159/000140715
12. Theocharis AD, Skandalis SS, Gialeli C, Karamanos NK. Extracellular matrix structure. Adv Drug Deliv Rev. 2016;97:4-27. PMID: 26562801 DOI: 10.1016/j.addr.2015.11.001
13. Mak KM, Mei R. Basement Membrane Type IV Collagen and Laminin: An Overview of Their Biology and Value as Fibrosis Biomarkers of Liver Disease. Anat Rec (Hoboken). 2017;300(8):1371-90. PMID: 28187500 DOI: 10.1002/ar.23567
14. Zhou S, Chen S, Pei YA, Pei M. Nidogen: A matrix protein with potential roles in musculoskeletal tissue regeneration. Genes Dis. 2022;9(3):598-609. PMID: 35782975 DOI: 10.1016/j.gendis.2021.03.004
15. Zbinden A, Layland SL, Urbanczyk M, Carvajal Berrio DA, Marzi J, Zauner M, et al. Nidogen-1 Mitigates Ischemia and Promotes Tissue Survival and Regeneration. Adv Sci (Weinh). 2021;8(4):2002500. PMID: 33643791 DOI: 10.1002/advs.202002500
16. Rosero Salazar DH, van Rheden REM, van Hulzen M, Carvajal Monroy PL, Wagener F, Von den Hoff JW. Fibrin with Laminin-Nidogen Reduces Fibrosis and Improves Soft Palate Regeneration Following Palatal Injury. Biomolecules. 2021;11(10):1547. PMID: 34680180 DOI: 10.3390/biom11101547
17. Huang QY, Lai XN, Qian XL, Lv LC, Li J, Duan J, et al. Cdc42: A Novel Regulator of Insulin Secretion and Diabetes-Associated Diseases. Int J Mol Sci. 2019;20. PMID: 30621321 DOI: 10.3390/ijms20010179
18. Umbayev B, Saliev T, Safarova Yantsen Y, Yermekova A, Olzhayev F, Bulanin D, et al. The Role of Cdc42 in the Insulin and Leptin Pathways Contributing to the Development of Age-Related Obesity. Nutrients. 2023;15(23):4964. PMID: 38068822 DOI: 10.3390/nu15234964
19. Ge J, Burnier L, Adamopoulou M, Kwa MQ, Schaks M, Rottner K, et al. RhoA, Rac1, and Cdc42 differentially regulate αSMA and collagen I expression in mesenchymal stem cells. J Biol Chem. 2018;293(24):9358-69. PMID: 29700112 DOI: 10.1074/jbc.RA117.001113
20. Lee NY, Suk KT. The Role of the Gut Microbiome in Liver Cirrhosis Treatment. Int J Mol Sci. 2020;22(1):199. PMID: 33379148 DOI: 10.3390/ijms22010199
21. Huang DQ, Terrault NA, Tacke F, Gluud LL, Arrese M, Bugianesi E, et al. Global epidemiology of cirrhosis-aetiology, trends and predictions. Nat Rev Gastroenterol Hepatol. 2023;20(6):388-98. PMID: 36977794 DOI: 10.1038/s41575-023-00759-2
22. Hasegawa S, Yoneda M, Kurita Y, Nogami A, Honda Y, Hosono K, et al. Cholestatic Liver Disease: Current Treatment Strategies and New Therapeutic Agents. Drugs. 2021;81(10):1181-92. PMID: 34142342 DOI: 10.1007/s40265-021-01545-7
23. Harrington C, Krishnan S, Mack CL, Cravedi P, Assis DN, Levitsky J. Noninvasive biomarkers for the diagnosis and management of autoimmune hepatitis. Hepatology. 2022;76(6):1862-79. PMID: 35611859 DOI: 10.1002/hep.32591
24. Sharma S, Khalili K, Nguyen GC. Non-invasive diagnosis of advanced fibrosis and cirrhosis. World J Gastroenterol. 2014;20(45):16820-30. PMID: 25492996 DOI: 10.3748/wjg.v20.i45.16820
25. Saikia P, Thangavadivel S, Medeiros CS, Lassance L, de Oliveira RC, Wilson SE. IL-1 and TGF-β Modulation of Epithelial Basement Membrane Components Perlecan and Nidogen Production by Corneal Stromal Cells. Invest Ophthalmol Vis Sci. 2018;59(13):5589-98. PMID: 30480706 DOI: 10.1167/iovs.18-25202
26. Pereira BA, Ritchie S, Chambers CR, Gordon KA, Magenau A, Murphy KJ, et al. Temporally resolved proteomics identifies nidogen-2 as a cotarget in pancreatic cancer that modulates fibrosis and therapy response. Sci Adv. 2024;10(27):eadl1197. PMID: 38959305 DOI: 10.1126/sciadv.adl1197
27. Hu X, Gan L, Tang Z, Lin R, Liang Z, Li F, et al. A Natural Small Molecule Mitigates Kidney Fibrosis by Targeting Cdc42-mediated GSK-3β/β-catenin Signaling. Adv Sci (Weinh). 2024;11(13):e2307850. PMID: 38240457 DOI: 10.1002/advs.202307850
28. Liu D, Tian X, Liu Y, Song H, Cheng X, Zhang X, et al. CREG ameliorates the phenotypic switching of cardiac fibroblasts after myocardial infarction via modulation of CDC42. Cell Death Dis. 2021;12(4):355. PMID: 33824277 DOI: 10.1038/s41419-021-03623-w
29. Choi SY, Chacon-Heszele MF, Huang L, McKenna S, Wilson FP, Zuo X, et al. Cdc42 Deficiency Causes Ciliary Abnormalities and Cystic Kidneys. J Am Soc of Nephrol. 2013;24(9):1435-50. PMID: 23766535 DOI: 10.1681/ASN.2012121236