Short-Term Western Diet Aggravates Non-Alcoholic Fatty Liver Disease (NAFLD) With Portal Hypertension in TGR(mREN2)27 Rats - PubMed (original) (raw)
doi: 10.3390/ijms21093308.
Robert Schierwagen 1, Frank Erhard Uschner 1, Sandra Torres 1, Olaf Tyc 1, Cristina Ortiz 1, Martin Schulz 1, Alexander Queck 1, Glen Kristiansen 2, Michael Bader 3, Tilman Sauerbruch 4, Ralf Weiskirchen 5, Thomas Walther 6 7, Jonel Trebicka 1 8 9 10, Sabine Klein 1
Affiliations
- PMID: 32392802
- PMCID: PMC7246932
- DOI: 10.3390/ijms21093308
Short-Term Western Diet Aggravates Non-Alcoholic Fatty Liver Disease (NAFLD) With Portal Hypertension in TGR(mREN2)27 Rats
Carla Cremonese et al. Int J Mol Sci. 2020.
Abstract
Non-alcoholic fatty liver disease (NAFLD) is gaining in importance and is linked to obesity. Especially, the development of fibrosis and portal hypertension in NAFLD patients requires treatment. Transgenic TGR(mREN2)27 rats overexpressing mouse renin spontaneously develop NAFLD with portal hypertension but without obesity. This study investigated the additional role of obesity in this model on the development of portal hypertension and fibrosis. Obesity was induced in twelve-week old TGR(mREN2)27 rats after receiving Western diet (WD) for two or four weeks. Liver fibrosis was assessed using standard techniques. Hepatic expression of transforming growth factor-β1 (TGF-β1), collagen type Iα1, α-smooth muscle actin, and the macrophage markers Emr1, as well as the chemoattractant Ccl2, interleukin-1β (IL1β) and tumor necrosis factor-α (TNFα) were analyzed. Assessment of portal and systemic hemodynamics was performed using the colored microsphere technique. As expected, WD induced obesity and liver fibrosis as confirmed by Sirius Red and Oil Red O staining. The expression of the monocyte-macrophage markers, Emr1, Ccl2, IL1β and TNFα were increased during feeding of WD, indicating infiltration of macrophages into the liver, even though this increase was statistically not significant for the EGF module-containing mucin-like receptor (Emr1) mRNA expression levels. Of note, portal pressure increased with the duration of WD compared to animals that received a normal chow. Besides obesity, WD feeding increased systemic vascular resistance reflecting systemic endothelial and splanchnic vascular dysfunction. We conclude that transgenic TGR(mREN2)27 rats are a suitable model to investigate NAFLD development with liver fibrosis and portal hypertension. Tendency towards elevated expression of Emr1 is associated with macrophage activity point to a significant role of macrophages in NAFLD pathogenesis, probably due to a shift of the renin-angiotensin system towards a higher activation of the classical pathway. The hepatic injury induced by WD in TGR(mREN2)27 rats is suitable to evaluate different stages of fibrosis and portal hypertension in NAFLD with obesity.
Keywords: ADGRE1; EMR1; F4/80; NAFLD; TGR(mREN2)27; Western diet; immunity; liver fibrosis; macrophage; portal hypertension.
Conflict of interest statement
The authors have no conflicts of interest.
Figures
Figure 1
Fibrosis in transgenic TGR(mREN2)27 rats after high fat Western diet. (A) Ten-week-old rats received either normal chow for 4 weeks, or normal chow for 2 weeks followed by Western diet for 2 weeks or Western diet for 4 weeks before hemodynamic measurements and harvesting of the organs. (B) Hepatic Sirius Red staining show more collagen deposition in TGR(mREN2)27 rats receiving WD compared to normal chow. (C) The densitometric analysis of (A) shows an increase of collagen positive areas after 2 and 4 weeks of WD feeding. (D). Collagen 1α1 (Col1α1) mRNA expression quantified by RT-qPCR is increased in livers of TGR(mREN2)27 rats after 2 weeks of WD. After 4 weeks, a decrease in Col1α1 levels are noticed, still being higher compared to rats receiving normal chow. (E) Livers of TGR(mREN2)27 rats receiving 2 or 4 weeks of WD express significantly more tumor growth factor-β (TGF-β) than TGR(mREN2)27 rats fed with normal chow. */*** p < 0.05/0.01 vs. normal chow, ## p < 0.01 vs. 2 weeks high fat Western diet.
Figure 2
α-smooth muscle actin (α-SMA) expression in transgenic TGR(mREN2)27 rats after high fat Western diet. (A) Hepatic α-SMA staining increased after 2 and 4 weeks of WD (left panel). (B) This effect is also seen in the quantification of α-SMA staining by densitometry. ** p < 0.01 vs. normal chow.
Figure 3
Hepatic assessment of macrophages and subgroup M1. (A) Hepatic frozen sections of TGR(mREN2)27 rats fed with normal chow, with 2 or 4 weeks of high fat Western diet were stained with the macrophage marker F4/80. Black arrow highlights positive cells. (B) Increased epidermal growth factor module-containing mucin-like receptor (Emr1) expression suggests increased numbers of macrophages after 4 weeks of WD feeding. (C) Increased expression of in CC-chemokine ligand 2 (Ccl2) was noticed after 4 weeks of WD feeding. (D,E) mRNA level of Il1β and TNFα confirmed the inflammatory effect after 2 and 4 weeks of WD feeding. */** p < 0.05/0.01 vs. normal chow.
Figure 4
Steatosis assessment in TGR(mREN2)27 rats. Effect of 2 weeks and 4 weeks of high fat diet in liver steatosis in TGR(mREN2)27 rats. (A) Representative Oil Red O staining and polarized light imaging (magnification ×100) of livers from TGR(mREN2)27 rats fed with normal chow, 2 and 4 weeks of high fat diet. (B) Levels of hepatic triglyceride content in TGR(mREN2)27 rats fed with normal chow and with 2 or 4 weeks of high fat diet. (C) Western blots of steatosis marker proteins (sterol regulatory element-binding protein 1c (Srebp-1c), fatty acid synthase (FAS)) and their quantification (D,E). The scale bar is 100 µm. Results are represented as mean ± SEM. For comparisons a two-tailed student´s unpaired t-test was performed; controls, n = 5 normal chow TGR(mREN2)27; n = 5 TGR(mREN2)27 with 2 weeks of high fat diet; n = 5 TGR(mREN2)27 with 4 weeks of high fat diet. All analyses were carried out using GraphPad Prism. * p < 0.05 vs. TGR(mREN2)27 rats fed with normal chow.
Figure 5
Hepatic and systemic effects of Western diet in TGR(mREN2)27 rats. (A) Portal pressure is increased after two and four weeks of WD. (B) Hepatic portal-vascular resistance increases after WD. (C) Mean arterial pressure shows no relevant alteration after WD feeding. (D) Cardiac output is reduced in rats receiving WD. (E) The renal arterial flow in TGR(mREN2)27 rats was investigated using the colored microsphere technique. Columns illustrate the renal arterial flow in mL/min/g kidney of rats. */** p < 0.05/0.01 vs. normal chow.
Figure 6
Western diet and RAS in TGR(mREN2)27 rats. Effect of high fat Western diet in RAS-system in TGR(mREN2)27 rats. (A) Increase in ACE and decrease in ACE2 levels. (B,C) Angiotensin-1-receptor and Mas-receptor were upregulated after two and four weeks of WD. */** p < 0.05/0.01 vs. normal chow.
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