Insights into hepatic and renal FXR/DDAH-1/eNOS pathway and
its role in the potential benefit of rosuvastatin and silymarin in
hepatic nephropathy
Yosra M. Magdy, Omnyah A. El-Kharashi, Dalia A.A. El-Waseef,
Enas S. Nabih, Abeer A. Abd El Samad
PII: S0014-4800(18)30259-4
DOI: doi:10.1016/j.yexmp.2018.10.004
Reference: YEXMP 4182
To appear in: Experimental and Molecular Pathology
Received date: 4 July 2018
Revised date: 5 September 2018
Accepted date: 6 October 2018
Please cite this article as: Yosra M. Magdy, Omnyah A. El-Kharashi, Dalia A.A. El￾Waseef, Enas S. Nabih, Abeer A. Abd El Samad , Insights into hepatic and renal FXR/
DDAH-1/eNOS pathway and its role in the potential benefit of rosuvastatin and silymarin
in hepatic nephropathy. Yexmp (2018), doi:10.1016/j.yexmp.2018.10.004
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Insights into Hepatic And Renal FXR/DDAH-1/eNOS Pathway and Its
Role in The Potential Benefit Of Rosuvastatin And Silymarin In Hepatic
Nephropathy
Yosra M. Magdya
, Omnyah A. El-Kharashia*
, Dalia A.A. El-Waseef b
, Enas S. Nabihc
Abeer A. Abd El Samad b
aDepartment of Clinical Pharmacology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
bDepartment of Histology and Cell Biology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
cDepartment of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Ain Shams University, Cairo,
Egypt
*corresponding author:[email protected]
Ain Shams University, Faculty of Medicine, Abbassia, Cairo, Egypt, P.O 38
Yosra M. Magdy; [email protected]
Omnyah A.El-Kharashi*;[email protected]
Dalia A.A. El-Waseef; [email protected]
Enas S. Nabih; [email protected]
Abeer A. Abd El Samad; [email protected]
ABSTRACT
Objectives: The repression of renal Farnesoid X Receptor (FXR) had been shown to result from
lack of bile acid production from cirrhotic liver. We hypothesized that silymarin and rosuvastatin
(Rvs) could have a hepatorenal therapeutic effects in hepatic nephropathy through induction of
FXR.
Methods: Forty two male Wistar rats were used; naïve (n=12); six of them were sacrificed after
4 weeks and six continued till the end of the experiment. Thirty rats were treated as follows: Rvs,
silymarin, thioacetamide (TAA), TAA+Rvs and TAA+silymarin. Liver and kidney function tests
as well as the renal and hepatic expression of transforming growth factor β1 (TGFβ1), FXR,
dimethylarginine dimethylaminohydrolase-1 (DDAH-1) and eNOS were performed. Histological
and immuno-histochemical studies of liver and kidney were also done.
Results: TAA-inducted liver cirrhosis was associated with significant deterioration of liver and
renal functions together with increasing expression of hepatic and renal TGFβ1 and decreasing
expression of hepatic and renal FXR, DDAH-1 and eNOS. Giving silymarin or Rvs induced
hepatic and renal improvement which was evidenced biochemically and histologically.
Significant positive correlation was detected between all the investigated biomarkers except for
the correlation between FXR and TGFβ1 which was negative.
Conclusions: In conclusion, liver cirrhosis is associated with deterioration of renal functions.
Silymarin and Rvs have a potential hepatorenal therapeutic benefit through simultaneous
enhancement of FXR/DDAH-1/eNOS pathway in both organs.
Keywords: Cirrhosis; DDAH-1; FXR; Kidney; Rosuvastatin; Silymarin.
I. Introduction
Liver cirrhosis has been associated with alterations in renal functions. One of the major
regulators in the development of liver cirrhosis and the associated renal impairment is nitric
oxide (NO). NO is produced from endothelial NO synthase (eNOS) enzyme and had been found
to be deficient in chronic renal failure patients (Passauer et al., 2005).
Asymmetric dimethylarginine (ADMA) is an endogenous NO inhibitor and higher plasma levels
of ADMA were detected in cirrhotic patients. It has been found that there is a close correlation
between ADMA plasma levels and the degree of hepatic dysfunction (Trauner et al., 2011).
Oxidative stress and inflammation are found to be responsible for increased synthesis and/or
inhibition of catabolism of ADMA (Kwaśny-Krochin 2012, cichoz-Lach and Michalak2014)
Additionally, Carello et al., 2006 found that ADMA elicits contractile effects on human renal
arteries and increased plasma levels of ADMA had been associated with a decrease in renal
plasma flow, increase in renovascular resistance, renal oxidative stress, endothelial dysfunction,
and induction of glomerular and vascular fibrosis through a mechanism involving collagen and
transforming growth factor β1 (TGFβ1) synthesis (Mihout etal. 2011).
Regarding the metabolism of ADMA, it is performed by dimethylarginine
dimethylaminohydrolases (DDAHs) which are expressed as type 1 and 2 isoforms and are
widely distributed in various organs and tissues, including the liver (Mookerjee et al. 2015) and
kidneys (Onozato et al., 2008). DDAH-1 was defined as a Farnesoid X receptor (FXR) target
gene, synthetic FXR agonist increased hepatic DDAH-1 gene expression (Hu et al., 2012).
The FXR is part of a family of nuclear hormone receptors that modulates the transcription of
many inflammatory and cell-cycle control genes and is particularly abundant in the kidney and
liver (Ali et al., 2015). Because FXR is the receptor of bile acids and is highly abundant in the
kidney, it was anticipated that FXR may share in the regulation of renal water and sodium
homeostasis and is involved in the development of fluid retention in liver cirrhosis. Both FXR
endogenous agonist (bile acid) and synthetic ligand (GW4064) induce FXR mRNA expression in
the kidney and in cultured renal collecting duct cells (Zhang et al., 2014).
Statins are recognized by their nephroprotective effect (Chmielewski et al., 2002). The hepato￾and nephroprotective effects of silymarin are also well studied (Alqasoumi, 2014, Mohamed et
al., 2018). Several studies documented increasing expression of FXR by statins (Byun et al.,
2014& (Lu et al., 2004 & Shimizu et al., 2014) and by silymarin (Gu et al, 2016).
We aimed in this study to investigate the involvement of FXR/DDHA-1/eNOS pathway in the
pathogenesis of hepatic nephropathy and whether silymarin and/or Rvs could improve hepatic
nephropathy through this pathway.
II. MATERIAL AND METHODS
II.1. drugs and chemicals:
Rosuvastatin (Rvs) (Glenmark Generics Ltd, India), Silymarin (Eva Pharma, Egypt); it is a
mixture of flavonolignans extracted from the milk thistle (Silybum marianum Gaertneri). The
main component of silymarin is silibinin (in a 50:50 mixture of Silybin A and Silybin B); the
remaining components are silydianin, silycristin, isosilybin A, isosilybin B, isosilycristin, and
taxifolin. Thioacetamide (TAA) (Sigma Aldrich Chemicals, Germany) all are dissolved in
saline.
II.2. Animals and grouping:
The protocol was approved by institutional ethical committee, animal care and the time of
sacrifice following the principles of the U.K. Animals Act, 1986 and associated guidelines, EU
Directive 2010/63/EU for animal experiments. The study design is presented in graph (1).
Male Wistar rats (n=42) of weight 150-200g, the experiment duration was 12 weeks. Rats were
allowed one week for acclimatization
The rats were divided into 2 groups: Naive (n=12) and treated groups:
 Control naïve group (n=12); after 4 weeks, 6 rats were scarified and used as control group
for Rvs and silymarin groups. Six rats had continued till the end of the experiment.
 Treated groups:
 Rosuvastatin (Rvs) group (n=6): Rvs (5 mg/kg/day/4weeks p.o) (Shirin et al.,
2013).
 Silymarin group (n=6): silymarin (50 mg/kg/day/4 weeks p.o) (Muriel et al.,
2005).
 TAA group (n=18): TAA was administrated for 12 weeks. This group was further
subdivided into 3 equal subgroups:
o TAA control (untreated)
o TAA-Rvs: received Rvs (5 mg/kg/day/4weeks p.o) in the last four weeks of the
experiment.
o TAA-Silymarin: received silymarin (50 mg/kg/day/4weeks p.o) in the last four
weeks of the experiment
II.3. Induction of liver cirrhosis:
Liver cirrhosis was induced by administration of TAA 200 mg/kg ip twice weekly for 12 weeks
and the rats were sacrificed by decapitation at 60 h after the last TAA (Mohamed et al., 2018)
II.4. Biochemical assays:
II.4.1. Samples processing for biochemical assays
II.4.1.1. Serum samples
Separated serum and collected 24 hours urine were stored at -80ºC until used. Serum was used in
the determination of alanine transaminase (ALT), aspartate transaminase (AST), alkaline
phosphatase (ALP), total and direct bilirubin, urea, creatinine and uric acid. Sodium was
determined in serum using TECO Diagnostics kit, Anaheim, CA, USA. 24 hours urine was
collected by metabolic cage according to the method of Hamed et al. (2017) and was used in the
measurement of microalbumin and creatinine used in the calculation of glomerular filtration rate
(GFR).
II.4.1.2. Tissue samples
The expression of TGFβ1, FXR, DDAH-1 and eNOS was assessed in liver and kidney by real
time PCR. Total RNA was purified from liver and kidney tissues using RNeasy mini Kit
(Qiagen, Hiden, Germany). One-step RT-PCR was done using LightCycler-RNA amplification
kit SYBR Green I (Roche Diagnostics, Mannheim, Germany). The final volume of each reaction
mixture was 20μl prepared according to the manufacturer’s instructions and containing 1μl RNA
and 2μl primer mix of either TGFβ1, FXR, DDAH-1 or eNOS forward and reverse primers (0.5
µM each). Primer sequences of TGFβ1 were: Forward, 5’-TGCTTCAGCTCCACAGAGAA-3’
and Reverse, 5’- TGGTTGTAGAGGGCAAGGAC-3’, GenBank: NM_021578.2, FXR:
Forward, 5′- CCAGGTCTCACTCAAGAATC-3′ and Reverse, 5′-
GTCCCTAGCTCAGTTGACAA-3′, GenBank: NM_021745.1, DDAH-1: Forward, 5′-
AGGACAAATCAACGAGGTGC-3′ and Reverse, 5′-TTTGC GCTTTCTGGGTACTC-3′,
GenBank: NM_022297.2, eNOS: Forward: 5′-TATTTGATGCTCGGGACTGC-3′ and Reverse,
5′-AAGATTGCCTCGGTTTGTTG-3’, GenBank: NM_021838.2. Primer sequences of the
reference gene (β-actin) were: Forward, 5’-AGATTACTGCCCTGGCTCCT-3’; Reverse 5’-
ACATCTGCTGGAAGGTGGAC-3’, GenBank: NM_031144.3. The reverse transcription,
denaturation and amplification were performed according the kit-based system protocol. The
expression of the target genes is presented based on RQ, ΔΔCt & ΔCt values.
II.5. Histological studies
At the end of the experiment, the right lobe of liver and the kidney of all rats were extracted
immediately. The right lobe of the liver was divided into two halves. One half of the liver and the
kidney were processed for light microscopy. The other half of the liver was processed for
electron microscopic examination.
II.5.1. Light microscopic (LM) study:
The liver specimens were cut into thin slices and the kidney was longitudinally divided into two
halves. Specimens were fixed in 10% neutral buffered formalin then processed to get paraffin
blocks for LM. The paraffin blocks were cut into five micron-thick sections for H&E and
Mallory’s trichrome stains. Immunostaining was done using an avidin biotin-peroxidase
technique for detection of α-SMA antibody (Lab vision, CA, USA) to assess liver and kidney
fibrosis at a dilution of 1:800 for one and half hour and proliferating cell nuclear antigen (PCNA)
antibody (Lab vision, CA, USA) in liver at a dilution of 1:200-400 for one and half hour was
done to assess proliferation of hepatocytes. The reaction was developed with DAB solution
(DAKO, Denmark) for 10 min. Finally, the slides were counterstained with Mayer’s
hematoxylin. Positive controls using smooth muscle cells (SMCs) and small intestine
respectively were prepared according to the same protocol. Negative controls were processed by
the same steps, except for the use of the primary antibody (Suvarna et al, 2013).
II.5.2. Transmission electron microscopic (TEM) study:
Liver specimen was cut into small parts (1 mm3) and fixed in 2.5% glutaraldehyde to be
prepared for TEM study. The specimens were processed and semi-thin sections (1µm) were cut
and stained with toluidine blue stain. Ultrathin sections (80 nm) were stained with uranyl acetate
and lead citrate and examined by transmission electron microscope (Joel, Japan) at the EM unit,
Faculty of Science, Ain Shams University (Suvarna et al, 2013).
II.5.3. Morphometric study:
Study was done using the image analyzer computer system Leica Qwin 500, UK to measure the
area % of collagen fibers (×40 power lens) and α-SMA in liver (×20 power lens) and kidney
(×40 power lens) as well as PCNA positive immune-reaction optic density (×20 power lens) in
liver. All parameters were measured in five non-overlapping fields of two serial sections from all
animals.
II.6. Statistical analysis:
Was carried with Graphpad prism, software program, version 5.0 (2007) (Inc., CA, USA).
Statistical difference among groups was determined using ANOVA followed by post hoc “Tukey
Test”. Correlation between different variables was performed by Pearson. Sample size was
determined using GraphPad StatMate, software program, Version 1.01i Jan. 16, 1998.
III. RESULTS
III.1. Biochemical parameters:
TAA produced significant deterioration of liver functions as evidenced by the results of liver
function tests that showed increasing levels of ALT, AST, bilirubin and ALP compared to
control group. Also, renal functions were deteriorated by TAA as evidenced by the results of
renal function tests that showed increasing creatinine, urea, uric acid and microalbumin levels
and decreasing sodium and GFR compared to control group. Although both liver and renal
functions were significantly improved by Rvs and silymarin, we found a significant difference
between both treated groups in favor of silymarin, Table (1). Moreover, TAA-Rvs and TAA￾silymarin treated groups significantly increased the urine volume compared to control and TAA￾treated groups. Giving control rats Rvs and silymarin for four weeks revealed significant increase
of the hepatic and renal expression of FXR compared to control untreated rats, Table (2).
Regarding the expression of hepatic and renal FXR, DDAH-1 and eNOS, the TAA-treated group
showed significantly lower levels of expression compared to control group. Both Rvs and
silymarin increased hepatic and renal expression of FXR, DDAH-1 and eNOS with no
statistically significant difference between both treated groups. When we evaluated the effect of
TAA on the hepatic and renal expression of TGFβ1, we detected a significantly higher
expression of TGFβ1 compared to control group. Both Rvs and silymarin decreased the hepatic
and renal TGFβ1 expression, Table (3).
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The correlation coefficients among the investigated biomarkers in treated groups for 12 weeks
revealed a negative association between the expression of hepatic TGFβ1 and hepatic FXR. On
the other hand, renal FXR expression revealed strong positive correlation with hepatic FXR,
renal DDAH-1 and renal eNOS expression. Renal DDAH-1 also showed strong positive
correlation with renal eNOS, Graphs (2-7). In TAA-Rvs and TAA-silymarin treated groups,
there was a week negative correlation between renal expression of FXR and TGFβ1 and this was
confirmed by calculating the mean % changes between them and TAA control group for these
biomarkers. By calculating the mean % changes of both parameters, we found that Rvs produced
+ 671% for renal FXR and -96% for TGFβ1 and silymarin produced +563% for renal FXR and -
69% for TGFβ1.
III.2. Histological results:
III.2.1. Results of liver:
III.2.1.1. H&E:
The control naive group showed the architecture of classic hepatic lobules (Fig.1A&B) which
lost in TAA control (untreated) group that showed cirrhotic nodules, thick fibrous tissue septa
with hemosiderin-engulfing cells and inflammatory cellular infiltration in between the nodules
(Fig.1C& Fig.1D). Most of the hepatocytes appeared with vacuolated cytoplasm with faint
vesicular nuclei, while others appeared having small pyknotic nuclei. In TAA-Rvs and TAA￾silymarin groups, areas of dense connective tissue from which incomplete septa arose.
Inflammatory cellular infiltration was seen in some septa but fewer than that in TAA untreated
group (Fig.1E&Fig 1G). In treated groups, hepatocytes were seen vacuolated with dense nuclei
while other hepatocytes appeared with central vesicular nuclei and acidophilic cytoplasm. TAA￾silymarin group showed more significant improvement than TAA-Rvs group than (Fig.1F& Fig
1H),
III.2.1.2. Mallory’s trichrome stain:
The TAA control (untreated) group showed increased the amount of collagen fibers especially in
the thick connective tissue septa surrounding the hepatic nodules (Fig.2B) in comparison to
control naïve group (Fig.2A). Both TAA-Rvs and TAA-silymarin treated groups showed a
decreased amount of collagen fibers in thin connective tissue septa between hepatic nodules
(Fig.2C& Fig.2D). Morphometric and statistical study revealed significant increase in the mean
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area percentage (MAP) of collagen fibers in TAA untreated group. Both TAA-Rvs and TAA -
silymarin groups significantly decreased the MAP of collagen fibers (Table 4).
III.2.1.3. α-SMA immune-reaction:
The control naïve group showed the classic immune-reaction for α-SMA (Fig.3A). TAA control
(untreated) group showed cells with positive immune-reaction for α-SMA in-between
hepatocytes, in the wall of blood vessels in the portal tracts and in the connective tissue septa
with a significant increase in the MAP of α-SMA immune-reaction (Fig.3B and Table, 4). Both
TAA- Rvs and silymarin groups showed an apparent decrease in α-SMA positive immune￾reaction in the cells in-between hepatocytes as well as in the connective tissue septa with a
significant decrease in the MAP of α-SMA immune-reaction compared to TAA control
(untreated) group (Fig.3C&Fig.3D and Table, 4).
III.2.1.4. PCNA immune-reaction:
The control naïve group showed the classic PCNA positive immune-reaction in their nuclei
(Fig.4A). In TAA untreated group, most of hepatocytes showed deep PCNA positive nuclei.
Some nuclei were seen enlarged with dense immune-reaction with a significant increase in mean
optical density (MOD) of PCNA positive nuclei (Fig.4B and Table 3). Both TAA-Rvs and TAA
-silymarin groups showed apparently decreased number and size of PCNA positive nuclei of
hepatocytes with a significant decrease in MOD of PCNA positive nuclei. There was a
significant difference between both groups favor TAA silymarin group (Fig.4C& Fig.4D and
Table 4).
III.2.1.5. TEM:
The control naïve group showed the classic hepatic structure (Figs.5A&5B). In TAA control
(untreated) group, the nuclei appeared with few chromatin, the mitochondria were electron￾lucent, few rER and condensed glycogen granule. Many lipid droplets with different sizes were
seen in the cytoplasm of hepatocytes. In addition, intra-nuclear fat droplets were sometimes seen
in hepatocytes. Collagen bundles appeared in-between hepatocytes near myofibroblast cells
(Figs.5C&5D). In both TAA-Rvs and TAA-silymarin groups, hepatocytes appeared with
euochromatic nuclei. Some lipid droplets were seen in the cytoplasm of hepatocytes (Fig.5E&
Fig.5F). In TAA-silymarin group, glycogen rosettes were seen in the cytoplasm of hepatocytes
(Fig.5F).
The control naïve group showed the classic structure of the kidney (Fig.6A& Fig.6B). TAA
control (untreated) group showed congestion of the glomerular capillaries and some blood
vessels in the cortex. Cells of PCT and DCT were seen with vacuolated cytoplasm. Some PCT
appeared having detached cells in their lumen. The wall of some arterioles in the renal cortex
appeared thickened with vacuolated SMCs (Fig.6C). The renal medulla showed markedly
vacuolated tubular cells and most of the tubules appear dilated. Dilated congested vasa recta and
extravasated RBCs in the renal interstitium were frequently seen (Fig.6D). TAA-Rvs group
showed renal glomeruli almost comparable to control group. Most of the cells lining PCT and
DCT showed some cytoplasmic vacuolations. Few congested blood vessels were also seen in the
renal cortex (Fig.6E). The renal medulla showed vacuolated tubular cells. Some tubules
appeared dilated. Some extravasated RBCs were seen in the renal interstitium as well as
congested vasa recta (Fig.6F). Whereas, TAA-silymarin treated group showed glomeruli with
congested capillaries and some congested blood vessels were also seen. Few cytoplasmic
vacuolations were observed in most of the cells lining PCT and DCT in the renal cortex
(Fig.6G). The renal medulla showed vacuolated tubular cells, but less than that in TAA control
(untreated) and, TAA-Rvs groups. Some tubules appeared dilated. Some extravasated RBCs
were seen in the renal interstitium with congested vasa recta (Fig.6H).
III.2.2.2. Mallory’s trichrome stain:
TAA control (untreated) group showed increased amount of collagen fibers around Bowman’s
capsule and renal tubules in the cortex. In addition, thick and wavy collagen bundles were seen
in the interstitium with a significant increase in MAP of collagen fibers in comparison to control
naïve group (Fig.7A & Fig.7B and Table 4). TAA-Rvs and TAA-silymarin groups showed few
collagen fibers around Bowman’s capsules and renal tubules in the cortex with significant
decrease in MAP of collagen fibers (Figs.7C & 7D and Table 5).
III.2.2.3. α-SMA immune-reaction:
TAA control (untreated) group showed positive immune-reaction for α-SMA in cells present
inside the renal corpuscles and in the interstitium around renal tubules in the cortex. Positive
immune-reaction was also seen in SMCs of the wall of blood vessels with a significant increase
in MAP of α-SMA immune-reaction in comparison to control naïve group (Fig.8A& Fig.8B).
TAA-Rvs group showed positive immune-reaction for α-SMA in cells present inside the renal
corpuscles and around the renal tubules in the cortex as well as in SMCs of the wall of blood
vessels (Fig.8C). On the other hand, TAA-silymarin group showed negative immune-reaction for
α-SMA in most of the renal corpuscles. Minimal positive immune-reaction was seen in cells
around renal tubules and positive immune-reaction was seen in SMCs of the wall of blood
vessels (Fig.8D). There is a significant decrease in MAP of collagen fibers in TAA-Rvs and in
TAA-silymarin groups (table 5).
IV. Discussion
This study reports novel findings regarding the link between hepatic cirrhosis and deterioration
of renal functions through FXR. We demonstrated that both Rvs and silymarin prevented liver
cirrhosis through FXR/DDAH-1/eNOS pathway. Moreover, they prevented renal impairment
that could associate liver cirrhosis through the same pathway. Although both drugs were
effective, however, silymarin induced better hepatic and renal effects compared to Rvs.
Patients with liver cirrhosis usually suffer from renal function deterioration which indicates a
link between these two organs (Forrest et al., 1996). It has been also reported that nonalcoholic
fatty liver plays a role in the development of chronic kidney disease (Galkina and ley, 2006)
(Natarajan et al., 2006). Although of suggestions that mediators such as nitric oxide play a role in
the development of renal failure in this situation, other mechanisms underlying this process are
not well understood yet. On the other side, renal dysfunction in patients with liver cirrhosis is
characterized by increased sodium and water retention leading to ascites, renal vasoconstriction
with decreased glomerular filtration rate which can lead to worsening of the clinical picture of
cirrhotic patients (Pipili and Cholongitas, 2014). Therefore, we conducted this study to compare
the potential therapeutic effect of silymarin as one of the well-known hepatoprotective drugs and
Rvs in a rat model of liver cirrhosis.
We induced liver cirrhosis by TAA being one of the experimental tools widely used to identify
hepatoprotective agents. We assessed the hepatic expression of TGFβ1 being a key regulator in
chronic liver disease and contributing to all stages of disease progression from initial liver injury
through inflammation and fibrosis to cirrhosis (Matsuzaki, 2009). The TAA-treated group
showed increasing TGFβ1 expression which was associated with deterioration of liver functions
evidenced by significant elevation of ALT, AST, bilirubin and ALP levels comparative to
control group. These results were in agreement with those of Reif et al. (2004) who found that
TAA-induced liver cirrhosis is considered to be a typical model because it mimics many aspects
of human liver diseases. Regarding our results concerning improvement of liver functions by
Rvs, they were in agreement with Seif el-din et al. (2015) who demonstrated that Rvs reduced
TGFβ1 in rats fed a high fat diet as an animal model of non-alcoholic fatty liver disease. By
comparing the hepatic effect of silymarin and Rvs, we found that both drugs improved liver
functions but silymarin gave better results. In animal experiments, silymarin was proved to have
protective effects on rat liver against hepatotoxicity induced by thioacetamide, acute ethanol
intoxication and carbon tetrachloride. It has been also found that treatment with ethanolic extract
of silymarin seed significantly declined the rats’ liver enzymes in the rat model of carbon
tetrachloride-induced liver damage (Wen Wu et al., 2009). In agreement with our results, shaker
et al. (2010) and Zhang et al. (2013) stated that silymarin has been introduced fairly as a
hepatoprotective agent and has been described to possess antioxidant, immunomodulatory,
antiproliferative, antifibrotic, and antiviral activities. Moreover, the study done by Saller et al.
(2007) found that silymarin inhibited the expression of many proinflammatory cytokines
resulting into reduction of the ALT and AST levels. Interestingly, the in vitro study done by
Morishima et al. (2010) on hepatitis C virus cell line showed that silymarin was able to inhibit T￾cell proliferation and proinflammatory cytokines secretion in a dose dependent manner.
Histological examination in H&E stained liver sections showed the presence of cirrhotic nodules,
increased amount of collagen fibers and inflammatory cellular infiltration. Similarly, Galkina
and Ley. (2006) and Natarajan et al. (2006) reported that administration of TAA resulted in liver
cirrhosis and fatty changes.
Ultrastructurally, hepatocytes showed abnormal organelles, accumulated glycogen granules
together with cytoplasmic fat droplets and occasional intranuclear lipid droplets. This could be
explained by some authors who reported that nuclear lipid droplets constitute specific
subdomains of the nuclear compartment probably involved in nuclear lipid homeostasis. They
suggested that nuclear lipid droplets may represent a source of rapidly available fatty acids for
nuclear membranes. They added that cytoplasmic lipid droplets are formed at the membrane of
endoplasmic reticulum, which is continuous with the nuclear envelope. It could be therefore
suggested that nuclear lipid droplets are actually cytoplasmic droplets trapped within an
invagination of the nuclear envelope (Uzbekov and Roingeard, 2013) and (Robert et al., 2016).
Other authors reported that neutral lipids are involved in many cellular processes and they are
stored as lipid droplets. Those are mainly cytosolic along with a small nuclear population.
Nuclear lipid droplets could be involved in nuclear-lipid homeostasis and could represent an
alternative source for providing fatty acids and cholesterol to membranes, signaling paths, and
transcription factors in the nucleus (Lagrutta et al., 2017) and (Layerenza et al., 2013).
Mallory stained sections showed significant increase in collagen fiber deposition around the
hepatic nodules together with significant increase in positive immune-reaction to α-SMA in
TAA-treated group. This was in accordance with previous studies which reported that
myofibroblasts which were developed form activated hepatic stellate cells or perivascular
fibroblast were the main cells producing the extracellular connective tissue (Schuppan et al.,
2003).
Concerning proliferation of hepatocytes, we detected a significant increase in optical density of
PCNA positive nuclei in TAA-treated group. This was in agreement with some investigators who
mentioned that in liver of control animals few hepatocytes showed positive immune expression
for PCNA, while increased expression was noticed in TAA-treated animals. This could be
attributed to response of hepatocytes to liver damage (Tousson et al., 2014).
Histologically, both Rvs and silymarin treated groups showed some hepatocytes with acidophilic
cytoplasm and central vesicular nuclei, while other hepatocytes were seen vacuolated. No
inflammatory cells could be noticed. This was explained by some authors who reported that
statins could reduce proinflammatory cytokines (Link et al., 2006). Rousuvastatin also decreased
endothelial dysfunction in liver cirrhosis (Abraldes et al., 2007) (Moreno et al., 2009). On the
other hand, it was previously reported that silymarin produced a significant improvement in the
structural, functional and architectural properties in cirrhotic livers (Salama et al., 2012).
Mechanism of action of silymarin was described by some authors who reported that it has
antioxidant effects and acts as a membrane stabilizer. It also promotes regeneration of
hepatocytes, decreases inflammation and prevents liver cirrhosis (Feher and Lenqyel, 2012)
Moreover, silymarin, was reported to prevent binding of hepatotoxin to hepatocyte membrane, in
addition to stimulation of rRNA polymerase and increasing protein formation (Dixita et al.,
2007). All these effects might be the cause of improvement of histological structure of the liver
and regeneration of hepatocyte observed in this study.
It has been reported that TGFβ1 is secreted form renal cells and cells infiltrating renal
interstitium (Border and Noble,1998) and is considered a key mediator in the occurrence of renal
fibrosis and inflammation (Lan and Chung, 2012). Our study showed a deterioration of renal
functions and increasing renal expression of TGFβ1 accompanying induction of liver cirrhosis by
TAA. Our findings were in agreement with the reports of Natarajan et al. (2006) who detected a
significant deterioration of renal functions by TAA evidenced by elevation of renal oxidative
stress markers and renal mitochondrial dysfunction.
Histologically, the TAA-treated rats showed kidney congestion of glomerular capillaries,
vacuolated tubular cells and thickening of the wall of some blood vessels were noticed. Dilated
tubules were also seen in the renal medulla. Similarly Neveen and Mahmoud. (2006) reported
that in chronic renal disease induced by liver cirrhosis, cells of convoluted tubules were
disturbed and swollen, some glomeruli were atrophied and hemorrhage was seen around blood
vessels. In the current study, both Rvs and silymarin treated groups, cytoplasmic vacuolation and
dilated tubules were still present. Significant increase in the mean area percentage of collagen
fiber deposition was noticed in renal interstitium in TAA-treated group together with significant
increase in positive immune reaction for α-SMA. Similarly Liu, et al., 2015 noticed increase
expression of α-SMA positive myofibroblasts in the renal interstitium. Moreover, TGFB1 could
increase secretion of collagen fiber deposition. TGFβ1 also inhibited degradation of extracellular
matrix (Border and Noble,1998) and caused tubular epithelial-mesenchymal transition of renal
epithelial cells to myofiobroblast cells which is responsible for collagen deposition in the renal
interstitium (Lanju 2003). Other authors reported that when the kidney is exposed to injury,
pericytes which surround the endothelial cells of blood capillaries detach and proliferate.
Pericytesare the main source of myofibroblasts that cause renal fibrosis (Lin et al., 2008).
We found that silymarin induced more improvement of renal functions compared to Rvs. Our
results were in agreement with previous studies. The effect of silymarin has been tested in
alloxan-induced diabetes mellitus models in rats. Alloxan produces reactive oxygen species
which injure renal tissue. Silymarin was administrated 20 days after 9 weeks treatment with
alloxan and it was effective on the renal tissue injuries. It has antioxidant effects via increase of
gene expression of antioxidant enzymes and a number of the most important protection
mechanisms against free radicals’ damage containing superoxide dismutase, glutathione
peroxidase, and catalase. Therefore, researchers concluded that silymarin could be used as a drug
for diabetic nephropathy therapy (Soto et al., 2010).
In the present study, silymarin induced moderate diuretic effect noticed by the increase in urine
volume/day. This is in agreement with de la Lastra et al.1991 who observed a diuretic behavior
for silymarin similar to potassium-sparing diuretics. This may add a therapeutic benefit to
silymarin in cirrhotic patient who are intolerable to the adverse effects of the potassium loosing
diuretics.
Histologically, both Rvs and silymarin treated groups showed a significant decrease in the mean
area percentage of collagen fiber deposition compared to TAA-treated group. It has been
reported that statins decrease proliferation of mesangial and vascular smooth muscle cells
(Campese et al., 2005), which might explain the decrease in the collagen fibers in the current
study in TAA Rvs-treated group.
FXR is one of nuclear hormone receptors and it has a rising role in the control of NO production
through targeting DDAH-1. Our results showed that TAA decreased expression of both hepatic
and renal FXR. Moreover, decrease hepatic and renal FXR was associated with decrease
expression of DDAH-1 and eNOS. Interestingly, administration of silymarin and Rvs was
associated by increasing expression of hepatic and renal FXR, DDAH1 and eNOS, decreasing
expression of TGFβ1 and hepatorenal therapeutic as evidenced by the results of hepatic and renal
function tests. The positive association detected through our results between hepatic and renal
FXR expression could explain the hepatorenal therapeutic effect of Rvs and silymarin through
increasing expression of FXR. FXR facilitates the tubular secretory mechanisms (Herman￾Edelstein et al., 2018), this may explain the moderate increase in urine volume by both drugs.
Naturally, the improvement of liver functions enhances bile acid synthesis which is considered
the main endogenous legend of renal FXR (Herman-Edelstein et al., 2018). The negative
association between hepatic FXR and TGFβ1 and the positive association between renal FXR
and both DDAH-1 and eNOS shed light on their therapeutic mechanisms. According to our
result, they protect the liver and the kidney through increasing expression of FXR which
increased expression of DDAH-1 resulting into increasing expression of eNOS. In agreement
with our results, researchers found that FXR was reduced in liver cirrhosis and that its activation
had a beneficial effect on liver cirrhosis as well as renal functions (Hu et al., 2012, Ding et al.,
2015). The weak negative correlation between renal FXR and renal TGFβ1 expression in TAA￾Rvs and TAA-silymarin treated groups could be explained by the enhancement effect of Rvs and
silymarin on FXR more than their inhibitory effect on TGFβ1, thus giving Rvs and silymarin the
priority in the management of hepatic nephropathy rather than primary renal affection.
Also, Hu et al, 2006 demonstrated that FXR agonists increased expression of DDAH1 gene with
subsequent reduction of its substrate ADMA. Since ADMA is a nitric oxide synthase inhibitor,
inhibition of ADMA by FXR agonists could play a protective role against the onset and
progression of renal diseases resulting from decrease NO production. Accordingly, it seems that
prevention of further liver deterioration together with renal functions preservation is the main
target in managing cirrhotic patients.
In conclusion, liver cirrhosis is associated with deterioration of renal functions. Silymarin and
Rvs have therapeutic effects through enhancement of the hepatic and renal FXR/DDAH-1/eNOS
pathway. However, silymarin was more effective than Rvs.
V. Ethics approval:
The entire experimental protocol was approved by institutional ethical committee and utmost care was
taken during the experimental procedure, as well as at the time of sacrifice following the principles of the
U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for
animal experiments.
VI. Availability of data and materials: PubMed.
VII. Consent for publication: Not applicable.
VIII. Competing interests: None.
IX. List of abbreviations:
ADMA: asymmetric dimethylarginine
ALK: Alkaline phosphatase
ALT: alaninetransaminase
AST: aspartate transaminase
CRD: Chronic Renal Disease
DDAH: dimethylarginine dimethylaminohydrolase￾ED: Endothelial dysfunction
e NOS:endothelial nitric oxide synthase
FXR: Farnesoid X receptor
GFR:Glomerular filtration rate
HFD: High fat diet
HSCs: Hepatic stellate cells
ip: Intraperitoneal
MAP: Mean area percentage
MOD: Mean optical density
NO: nitric oxide
OS: Oxidative stress.
PCNA: proliferating cell nuclear antigen
PCR: polymerase chain reaction
RT PCR: reverse transcripatase polymerase chain reaction
Rvs: Rosuvastatin
αSMA: α smooth muscle actin
SMCs: Smooth muscle cells
TGFβ1: Transforming growth factor β1.
X. Authors contributions:
 Yosra M. Magdy: Share in Idea, design, pharmacological work and writing the paper
manuscript.
 Omnyah A. El-Kharashi: Share in Idea, design, pharmacological work and writing the paper
manuscript.
 Dalia A.A. El-Waseef: Share in Idea, design, histological work and writing the paper
manuscript.
 Enas S. Nabih: Share in Idea, design, biochemical work and writing the paper manuscript.
 Abeer A. Abd El Samad: Share in Idea, design, histological work and writing the paper
manuscript.
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Fig. (1A): showing the architecture of classic hepatic
lobule with central vein (C) and portal tracts (P) at the
corners.
Group I (H&E × 100)
Fig. (1B): showing plates of hepatocytes around
central vein (C) with blood sinusoids (S) in-between.
Hepatocytes are polygonal in shape with central
vesicular nuclei and acidophilic cytoplasm.
Group I (H&E × 400
Fig. (1C): showing loss of the normal hepatic
architecture. Cirrhotic nodules appear with different
shapes and sizes. Thick fibrous tissue septa with
inflammatory cellular infiltration (→) are seen between
the nodules.
Group II (H&E × 100)
Fig. (1D): showing most of hepatocytes (→) with
vacuolated cytoplasm. Thick fibrous tissue septum is
seen between nodules with hemosiderin-engulfing
cells (▲).
Group II (H&E × 400)
Fig. (1E): showing areas of dense connective tissue (→)
from which incomplete C. T. septa (wavy arrow) arise
between nodules.
Group III (H&E × 100
Fig. (1F): showing thin connective tissue septa
between nodules. Some hepatocytes (↑) appear with
central vesicular nuclei and acidophilic cytoplasm and
other hepatocytes are vacuolated.
Group III (H&E × 400)
Fig. (1G): showing areas of dense connective tissue (→)
from which incomplete C. T. septa (wavy arrow) arise
between hepatic nodules.
Group IV (H&E × 100)
Fig. (1H): showing thin connective tissue septa
between hepatic nodules. Many hepatocytes are seen
vacuolated while other hepatocytes (→) appear with
central vesicular nuclei and acidophilic cytoplasm.
Group IV (H&E × 400)
2-Mallory’s Trichrome stain:
Fig. (2A): showing few collagen fibers around central
veins (→) and in portal tracts (▲).
Group I (Mallory’s trichrome × 100)
Fig. (2B): showing increase collagen fibers in thick septa (↑)
surrounding the hepatic nodules.
Group II (Mallory’s trichrome × 100)
Fig. (2C): showing decreased collagen fibers in C.T.
septa (→) between hepatic nodules.
Group III (Mallory’s trichrome × 100)
Fig. (2D): showing decreased collagen fibers (→) in the
incomplete septa between hepatic nodules.
Group IV (Mallory’s trichrome × 100)
3-α-SMA immune-reaction:
Fig. (3A): showing positive immune-reaction for α-
SMA in smooth muscle cells (→) in the wall of blood
vessel in portal tract.
Group I (Avidin Biotin Peroxidase for α-SMA ×
400)
Fig. (3B): showing cells with positive immune-reaction for
α-SMA in-between hepatocytes (▲), in the wall of blood
vessels in the portal tract (→) and in the CT septa (curved
arrow).
Group II (Avidin Biotin Peroxidase for α-SMA × 400)
Fig. (3C): showing an apparent decrease in α-SMA
positive immune-reaction in cells in-between
hepatocytes as well as in the connective tissue septa
(▲).
Group III (Avidin Biotin Peroxidase for α-SMA ×
400)
Fig. (3D): showing minimal positive immune reaction for α-
SMA (▲).
Group IV (Avidin Biotin Peroxidase for α-SMA ×
400)
4-PCNA immune-reaction:
Fig. (4A): showing few hepatocytes with faint PCNA
positive immune-reaction in their nuclei.
Group I (Avidin Biotin Peroxidase for PCNA × 400)
Fig. (4B): showing most of hepatocytes with deep PCNA
positive nuclei. Note the dense immune reaction and
increased size of some nuclei (▲).
Group II (Avidin Biotin Peroxidase for PCNA × 400)
Fig. (4C): showing apparently decreased number and
size of PCNA positive nuclei of hepatocytes. Some
hepatocytes are seen with dense immune-reaction
(▲). Group III (Avidin Biotin Peroxidase for PCNA
× 400)
Fig. (4D): showing apparently decrease number and size of
PCNA positive nuclei with some nuclei showing dense
immune-reaction (▲).
Group IV( Avidin Biotin Peroxidase for PCNA ×
400)
Fig. (5A): showing part of a nucleus (N) of a
hepatocyte, mitochondria (M) and rER (∆).
Group I (TEM × 4000
Fig. (5B): showing an Ito cell with lipid droplets (→) and
the surrounding hepatocytes.
Group I (TEM×1200
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Fig. (5C): showing nucleus (N) of hepatocyte with few
chromatin, electron-lucent mitochondria (M), few rER
(▲) and many lipid droplets (L). Note an intra-nuclear
lipid droplet (curved arrow) and condensed glycogen
granules in the cytoplasm.
Group II (TEM × 1500)
Fig. (5D): showing a myofibroblast cell (→) with elongated
nucleus. Collagen bundles are also seen (wavy arrow).
Group II (TEM × 2500)
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Fig. (5E): showing euochromatic nucleus (N) of
hepatocyte, mitochondria (M) and rER (▲). Few lipid
droplets (L) are seen.
Group III (TEM ×
2000)
Fig. (5F): showing nucleus (N) of hepatocyte, mitochondria
(M) and rER (▲).
Group IV (TEM × 2500)
Results of the kidney:
1-H&E:
Fig. (6A): showing the renal cortex in group I. A
glomerulus (G) is seen surrounded by Bowman’s capsule.
Proximal convoluted tubules (P) are seen with narrow
lumen and deep acidophilic pyramidal cell lining. Distal
convoluted tubules (D) appear with wide lumen and
acidophilic cubical cells.
Group I (H&E × 400)
Fig. (6B): showing the renal medulla in group I, formed of
cut sections in the collecting tubules and loops of Henle as
well as the vasa recta. Fig. (6C): showing markedly congested blood vessels (V).
An arteriole is seen with thickened wall (▲). Cells of PCT
(P) and DCT (D) are seen with vacuolated cytoplasm. Note
the congested glomerular capillaries (↑).
Group II (H&E × 400)
Fig. (6D): showing markedly vacuolated tubular cells. Most
of the tubules appear dilated. Extravasated RBCs (↑) and
congested vasa recta are seen.
Group II (H&E × 400)

Fig. (6E): showing a glomerulus (G) surrounded by
Bowman’s capsule. Some cytoplasmic vacuolations are
seen in most of the cells lining PCT (P) and DCT (D). A
congested blood vessel (V) is also seen.
Group III (H&E × 400)
Fig. (6F): showing vacuolated tubular cells, some tubules
appear dilated (T) and some extravasated RBCs (↑) are seen
in the interstitium. Note congested vasa recta.
Group III (H&E × 400)
Data are mean±SD of 6 rats per group. *
p<0.05 vs. control (naïve) group; #
p<0.05 vs. TTA control group, $
p<0.05 vs TAA-Rvs
treated group, ^
p<0.05 vs TAA-silymarin treated group, by One-way ANOVA with Tukey post-hoc test.
Table (4): Showing the mean±SD of area percentage of collagen fibers and α-SMA and the
optical density of PCNA positive nuclei in liver of different groups
Groups Area % of Collagen
Data are mean±SD of 6 rats per group. *
p<0.05 vs. control (naïve) group; #
p<0.05 vs. TTA
control group, $
p<0.05 vs TAA-Rvs treated group,
p<0.05 vs TAA-silymarin treated group by ZD4522
One-way ANOVA with Tukey post-hoc test.