Chenodeoxycholic acid alleviated the cyclosporine-induced nephrotoxicity by decreasing oxidative stress and suppressing renin-angiotensin system through AT2R and ACE2 mRNA upregulation in rats

Chenodeoxycholic acid alleviated the cyclosporine-induced nephrotoxicity by decreasing oxidative stress and suppressing renin-angiotensin system through AT2R and ACE2 mRNA upregulation in rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Chenodeoxycholic acid alleviated the cyclosporine-induced nephrotoxicity by decreasing oxidative stress and suppressing renin-angiotensin system through AT2R and ACE2 mRNA upregulation in rats Ilknur Bingul, Rivaze Kalayci, Merva Soluk Tekkesin, Vakur Olgac, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4635970/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Dec, 2024 Read the published version in Journal of Molecular Histology → Version 1 posted 9 You are reading this latest preprint version Abstract Objective Oxidative stress, inflammation and renin-angiotensin system (RAS) activation play an important role in the nephrotoxicity which is caused by the long-term use of the immunosuppressive drug cyclosporine (CsA). This study investigates whether chenodeoxycholic acid (CDCA), an endogenous farnesoid X receptor (FXR) agonist with antioxidant and anti-inflammatory effects, modulates CsA nephrotoxicity. Methods CsA (25 mg/kg/day; s.c.) was administered to rats for 12 days. CDCA (20 mg/kg/day; i.p.) injection was started 3 days before CsA and continued for 15 days. CDCA improved renal damage and function in CsA-administered rats. Renal function markers in serum, renal histology, oxidative stress, inflammation and RAS components were determined in kidney. Results CDCA reduced CsA-induced renal increases in NADPH oxidases 4 and NADPH oxidases 2 mRNA expressions, oxidative stress and inflammation. CDCA elevated renal FXR, small heterodimer partner-1, hypoxia-inducible factor and vascular endothelial growth factor and nuclear factor erythroid 2-related factor mRNA expressions in CsA rats. It prevents renin angiotensin system activation by reducing angiotensin II (Ang-II) levels in serum and upregulating renal mRNA expressions of Ang II type-II receptor (AT2R) and angiotensin converting enzyme 2 (ACE2), but not AT1R and ACE in CsA rats. Conclusions Our results indicate that CDCA may be a protective agent against CsA-nephrotoxicity by decreasing inflammation, oxidative stress and RAS activation via AT2R and ACE2 upregulations. Chenodeoxycholic acid Cyclosporine A Oxidative stress Nephrotoxicity Renin angiotensin system Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Cyclosporine A (CsA), an immunosuppressant agent, commonly used for prevention of organ rejection after transplantation surgeries, is also utilized for therapy of some autoimmune diseases. However, renal and vascular side effects commonly occur by long-term use of CsA. Proposed mechanisms include imbalance between vasoconstrictor and vasodilator substances, enhanced activation of inflammation, oxidative stress and the renin angiotensin system (RAS) (Yoon and Yang 2009 , Hoskowa et al. 2017). Animal studies indicated that CsA alters kidney function markers and histopathological findings along with oxidative stress and inflammation (Hagar et al. 2006 ; Chia et al. 2013 ; Tan et al. 2020 ; Nouri et al. 2022; Kalaycı et al. 2023 ). CsA caused endothelial dysfunction, disturbed vasodilation and vasoconstriction reactions and enhanced ROS formation in vascular system (8,9). Moreover, CsA was detected to stimulate renal and aortic RAS activation (Hu et al. 2002; Nishiyama et al. 2003 ; Bekpinar et al. 2019 ; Kalaycı et al. 2023 ). RAS takes role in blood pressure, water, electrolyte balance in the organism and it consists of two axes as classical and alternative axes. The components of classical RAS are angiotensin converting enzyme (ACE), angiotensin II (Ang-II) and Ang II type-1 receptor (AT1R). The main molecule in RAS is Ang-II and it increases the contractility of vessels by binding to the Ang-II type-1 receptor (AT1R), causing oxidative stress, inflammation, proliferative and fibrotic changes. Other receptor of Ang-II is the AngII type-2 receptor (AT2R). When Ang-II binds to AT2R, it exerts opposite effects (vasodilatory, anti-inflammatory, antiproliferative, antifibrotic). However, the affinity of Ang-II for AT2R is very low [12,13]. On the other hand, the alternative axis consists of ACE2, angiotensin-(1–7) [Ang-(1–7)] and Mas receptor (MasR). ACE2 is an enzyme that converts Ang-II to Ang-(1–7). Ang-(1–7) shows vasodilator, anti-inflammatory, antiproliferative and antifibrotic effects through MasR (Ma et al. 2022 ; Vargas Vargas et al. 2022 ). The farnesoid X receptor (FXR) is expressed to a significant extent in the liver, kidney and gastrointestinal tract and activated by bile acids or derivatives of bile acids (BAs). As activated by a ligand, FXR can bind to a DNA response element of multiple genes, including small heterodimer partner (SHP), and activates or represses their transcriptions (Han et al. 2018). FXR was reported to regulate several genes involved bile acid, glucose, lipid and intestinal flora metabolisms, inflammation and oxidative stress stress (Han et al., 2018; Zhang et al. 2020 ). Therefore, FXR agonists were reported to be effective in metabolic and cardiovascular disorders, hepatic steatosis and fibrosis/cirrhosis, acute and chronic renal injuries (Han et al. 2018; Zhang et al. 2020 ; Kim et al. 2023 ). Natural agonists of FXR are primary [including chenodeoxycholic acid (CDCA) and cholic acid (CA)] and secondary BAs [such as lithocholic acid (LCA) and deoxycholic acid (DCA)]. The order of their potency in activating FXR were detected as CDCA > DCA > LCA > CA. Apart from these natural agonists, synthetic agonists such as obeticholic acid and GW-4064 have been developed. These agonists are more powerful than BAs in activating FXR and they have been extensively utilized in both experimental and clinical studies compared to their natural agonists (Han et al.2018; Zhang et al. 2020 ). CDCA is the most potent natural FXR agonist. CDCA was reported to improve glucose tolerance and hepatic fat accumulation induced by high fat diet (Chen et al. 2017 ) and dinitrobenzene sulfonic acid (DNBS]-induced ulcerative colitis (Goyal et al. 2015 ) in rats. Moreover, CDCA reduced blood pressure, improved vascular relaxation and contraction responses of mesenteric arteries obtained from spontaneously hypertensive rats (Li et al. 2015 ). There are several reports investigating the effect of FXR agonists including CDCA on acute and chronic nephrotoxicity (Kim et al. 2023 ; Yang J et al. 2024 ). FXR agonists obeticholic acid and GW-4064 were reported to alleviate acute renal injury due to ischemia-reperfusion injury (Gai et al 2017 ), and cisplatin (Kim et al. 2022 ). Similarly, they alleviated renal damage in mice fed with high fat- high cholesterol diet (Wang et al. 2009 ) and uninephrectomized obese mice (Gai et al. 2016 ). Moreover, INT-767 (FXR/TGR5 agonist) also prevented kidney damage in STZ-induced diabetic mice, db/db mice and obese mice fed on high fat diet (Wang et al. 2018 ). Similarly, CDCA also inhibited the progression of diabetic nephropathy Kkay mice, an animal model of type 2 diabetes (Takebayashi and Inukai 2014 ) and prevented kidney injury induced by high fructose diet in rats (Hu et al. 2012 ). In the literature, no in vivo study reporting the effects of FXR agonists on CsA-mediated nephrotoxicity is noted but in mouse cut liver slides incubated with CsA, FXR and its target genes were found to be downregulated (Szalowska et al. 2013 ). For this reason, based on the antilipogenic, antioxidant, anti-inflammatory and antifibrotic properties of CDCA, it is conceivable that it may be effective against CsA-induced toxicity. Therefore, the outcomes of CDCA administration on CsA nephrotoxicity was investigated for the first time by determining oxidative stress, inflammation and classical and alternative axes of RAS in rats. Materials and Methods Chemicals CsA (Sandimmune) from Novartis Pharmaceutical (Switzerland), and CDCA (GP-7061) from Glentham Life Sciences (United Kingdom), and other laboratory chemicals from Sigma-Aldrich (USA) were utilized. Animals and experimental model Male Wistar rats (6–8 months old) weighing 300–320 g were used. They were supplied from Bezmialem University, Experimental Medical Research Institute. They were kept in stainless steel cages (3–4 rats per cage) under controlled temperature, humidity and 12 h light/dark cycles. No feed and water restrictions were applied to guinea pigs. The protocol for this study was reviewed and approved by the Bezmialem University Animal Care and Use Committee. Rats were divided into 4 groups randomly; a) Control (n = 7): Animals received standard rat chow and drinking water without restriction for 15 days. b) CDCA (n = 7): CDCA was dissolved freshly in dimethyl sulfoxide (DMSO) and injected to rats intraperitoneally (i.p.) at a dosage of 20 mg/kg daily for 15 days. c) CsA (n = 8): Animals received CsA at a dosage 25 mg/kg/day subcutaneously (s.c.) for 12 days. d) CDCA + CsA (n = 8): CDCA administration at a dosage 20 mg/kg/day (i.p.) to rats was started 3 days before CsA and continued with CsA at a dosage 25 mg/kg/day (s.c) for a total of 15 days. The duration and dose of CDCA [18,19] and CsA [4,5] administrations were adjusted according to available studies in literature. DMSO was also applied to control group according to experimental procedure as vehicle. Food and water intake and body weights were detected during the experiment. Rats from each group were transferred separately to metabolic cages to collect urine (24h) samples before last day of experimental protocol, and they were kept at -20 O C. Collecting of samples In the last stage of the experiment, rats were anesthetized by ketamine and xylazine HCl injections. Rats were sacrificed to collect whole blood through the cardiac puncture, then the sera were separated by the centrifugation at 1500g for 10 minutes. Kidneys were immediately removed. The cold phosphate buffered saline (PBS; 0.01 M, pH: 7.4) was utilized to prepare 10% homogenates (w/v), and then they were centrifuged at 600xg for 10 min at 4°C and supernatants were utilized for biochemical analyses. The antioxidant enzyme activities were also assessed in the postmitochondrial fraction, which was obtained by centrifuging of the supernatants at 10000xg for 20 min at 4 o C. All materials were stored at -80 o C until they were needed for analyses. The left kidney was fixed with formaldehyde for histopathological analyses. Urine samples were centrifuged at 1750xg for 10 min, supernatants were used for analyses. Kidney function evaluation The levels of blood urea nitrogen (BUN) and creatinine (Cr) were estimated with a Cobas-Integra 800 autoanalyser (Roche Diagnostics, Germany). This autoanalyser was also used for the determination of urine protein and urine Cr levels. Glomerul filtration rate was evaluated with Cr clearance test (CCr) and was computed with the formula: CCr (mL/min) = the ratio of urine Cr (mg/dL)x24h urine volume (mL) to serum Cr (mg/dL)x1440. The measurement of serum Ang-II and Ang (1–7) levels Ang-II (KTE101212, Abbkine, Inc. China) and Ang 1–7 (KTE101211, Abbkine, Inc. China) determinations were done with enzyme linked-immunosorbent assay (ELISA) kits. The evaluation of renal reactive oxygen species (ROS) and lipid peroxide levels Reactive oxygen species levels (ROS) generation was determined in kidney homogenates incubated with 100 µM 2′,7′-dichlorodihydrofluorescein diacetate (Wang and Joseph 1999 ). The fluorescence of the reaction was estimated with a Fluoroskan Ascent analyser from Thermo Scientific (USA), with an excitation of 485 nm and emission of 538 nm. Absorbances were given as relative fluorescent unit (RFU/mg protein). Renal thiobarbituric acid reacting substances (TBARS) levels were assessed in rats to evaluate lipid peroxidation (Buege and Aust 1978 ). Renal homogenates were incubated with Buege-Aust reagent in a boiling water bath. Then, the mixture was cooled and centrifuged at 1000xg for 10 min. Absorbances were recorded at 532 nm and computed with the molar extinction-coefficient value (1.56x10 − 5 M − 1 cm − 1 ) and expressed as pmol/mg protein. Renal superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activities SOD activity was measured according to spectrophotometric method described Mylorie et al. (Mylorie et al. 1986 ). Bovine SOD was used as standard. The difference in absorbances between 0 and 8 min of illumination was detected at 540 nm. Absorbances were calculated as U/mg protein. The cumene hydroperoxide was utilized as a substrate to estimate activity of GSH-PX and absorbances were given as nmol NADPH/min/mg protein (Lawrence and Burk 1976 ). Determination of renal tumor necrosis-alpha (TNF-α) levels and myeloperoxidase (MPO) activity TNF-α levels were measured using an ELISA kit (#MBS 2882073, MyBioSource, Inc., San Diego, USA), and results were expressed as pg/mL. MPO was assayed by determining H 2 0 2 dependent oxidation of o-dianisidine in the kidney homogenates. The molar extinction coefficient value (1.3×104 M − 1 cm − 1 ) of oxidised o-dianisidine was utilized for the calculation (Rachmilewitz et al. 1993 ). Results were given as nmol/min/mg protein. Determination of protein levels Protein levels were determined spectrophotometrically using bicinchoninic acid (Smith et al. 1985 ). mRNA expressions in renal tissue FXR, SHP-1, NADPH oxidases (NOX4, NOX2), nuclear factor erythroid 2-related factor (Nrf2), hypoxia-inducible factor 1-α (HIF-1α), vascular endothelial growth factor (VEGF), AT1R, AT2R, ACE and ACE2 mRNA expressions were determined in renal tissue. For this reason, kidney tissues were homogenized, total RNA was isolated with NucleoSpin RNA Isolation Kit (#740955) from Macherey-Nagel (Germany) and then cDNA synthesis with SCRIPT cDNA Synthesis Kit from Jena Bioscience, GmbH (Jena, Germany) were performed using 5 ng RNA template. Quantitative real time polymerase chain reaction (qRT-PCR) analyses were conducted with the primers pairs of forward and reverse acquired from LGC Biosearch Technologies (Denmark), as detailed in Table 1 , in a RT-PCR system (Biorad CFX Connect, California, USA). The 2 −ΔΔCt formula was utilized for computing the mRNAs expressions. Table 1 Primer sequences used for qRT-PCR analysis of target mRNA. FXR F: 5'-CATTACAACGCGCTCACCTG-3'; R: 5'- TTCCTTAGCCGGCAATGGTG-3' SHP-1 F: 5'-AGTCTTTCTGGAGCCTTGAGC-3' R: 5'-CAGGACTTCACACAATGCCC-3' NOX4 F: 5′-CGTCCACCGTTACCAGACAA-3′ R: 5′-TTGGCCTCTGCGTATTCGTT-3′ NOX2 F: 5’-TGGCGATCTCAGCAAAAGGTGG-3’ R: 5’-GTACTGTCCCACCTCCATCTTG-3’ Nrf2 F: 5’-GTGGATCTGTCAGCTACTCCC-3’ R: 5’-CTGGGAATATCCAGGGCAAGC-3’ HIF-1α F:5’-ATGTACCCTAACAAGCCGGG-3’ R: 5’-AAGCACGTCATAGGCGGTTT-3’ VEGF F:5’-GCACTGGACCCTGGTTTAC-3’ R:5’-GGGTCTCAATTGGACGGCAA-3’ AT1R F: 5'-TTCACCCTGCCTCAGGATCT-3' R: 5'-CCAGACCCACCAATCCATCC-3' AT2R F:5'-AGAAGGAATCCCTGGCAAGC-3' R: 5'-TAAGGCAATCCCAGCAGACC-3' ACE F: 5'-CGTCCACCGTTACCAGACAA-3' R: 5'-TTGGCCTCTGCGTATTCGTT-3' ACE2 F: 5'-GAATGCGACCATCAAGCGTC-3' R: 5'-CAAGCCCAGAGCCTACGATT-3' GAPDH F: 5′-CAGGG CTGCCTTCTCTTGTG-3′ R: 5′-AACTTGCCGTGGGTAGAG TC-3′ Histological analysis Kidney tissue was fixed with 10% buffered formalin solution and embedded in paraffin. Sections were cut at 5 µm and stained with hematoxylin–eosin (H&E) and then histopathological changes were examined by light microscopy. Renal damage was evaluated and scored conducted in accordance with the protocol outlined by Al-Rejaie et al ( 2012 ). Rabbit anti-4-hydroxynonenal (4-HNE) primary antibody (Catalog No. orb 100588; Biorbyt, Cambridgeshire, UK) and kidney sections were incubated, and renal 4-HNE were semiquantified by immunostaining. Additionally, positive control studies were conducted in sections of healthy human kidneys. The presence or absence of brown staining was considered indicative of a positive or negative result for each antibody, respectively. Digital photographs were assessed using the Olympus AnalySIS 5 image analysis program. Statistical analysis In this study, ‘Statistical Package for Social Sciences Program’ (21.0; SPSS Inc., Chicago, IL, USA) were utilized for statistical analysis. The values were given as mean ± standard error of the mean (SEM). Under parametric or non-parametric test conditions, mean values were compared between groups by One-way ANOVA test (post-hoc Tukey’s test) or Kruskal-Wallis test (post-hoc Mann Whitney-U test), respectively. A value of p < 0.05 was considered statistically significant. Results Body and kidney weights changes and kidney index Significant decreases in final body weights were found in CDCA (14.0%; p = 0.000), CsA (13.3%; p = 0.000) and CDCA + CsA (24.0%; p = 0.000) groups as compared to controls. In CDCA + CsA group, final body weights of rats are significantly lower than those of CsA-rats (p = 0.000). Kidney weights diminished significantly in CsA and CDCA + CsA groups (p = 0.005; p = 0.001, respectively), but kidney index values remained unchanged as compared to controls (Table 2 ). Table 2 The effect of chenodeoxycholic acid (CDCA) on body and kidney weights and indexes in cyclosporine (CsA)-treated rats (Means ± SEM) Control (n = 7) CDCA (n = 7) CsA (n = 8) CDCA + CsA (n = 8) Initial body weight (g) 322.8 ± 6.80 330.1 ± 2.39 331.9 ± 4.42 311.9 ± 6.74 Final body weight (g) 350.6 ± 6.87 301.6 ± 3.21 a 303.7 ± 7.10 a 266.5 ± 4.77 a,b Gain/15 days (g) 27.7 ± 2.80 -28.6 ± 3.91 a -28.1 ± 4.30 a -45,4 ± 4.38 a,b Kidney weight (g) 2.79 ± 0.08 2.67 ± 0.10 2.42 ± 0.04 a 2.33 ± 0.05 a Kidney index (%) 0.79 ± 0.02 0.89 ± 0.04 0.80 ± 0.01 0.88 ± 0.02 a p < 0.05 compared with the control group; b p < 0.05 compared with the CsA group. CDCA treatment alleviated disturbances in renal function markers in CsA-rats Serum BUN (p = 0.000), Cr (p = 0.000) and urinary protein levels (p = 0.037) and the ratio of urinary protein level to creatinine level (p = 0.002) elevated, but CCr values decreased significantly (p = 0.038) in CsA-applied rats. CDCA treatment decreased serum BUN (p = 0.01) and urinary protein levels (p = 0.012) and the ratio of urinary protein level to Cr level (p = 0.006) and increased CCr values (p = 0.036) in CsA-rats. Although serum Cr levels decreased by 18.5%, this change was not statistically significant when comparing the CDCA + CsA group to the CsA group (Table 3 ). Table 3 The effect of chenodeoxycholic acid (CDCA) on blood urea nitrogen (BUN), serum creatinine (Cr) and urine protein levels, creatinine clearance (CCr)values and daily urine volume in cyclosporine (CsA)-applied rats (Means ± SEM). Control (n = 7) CDCA (n = 7) CsA (n = 8) CDCA + CsA (n = 8) BUN (mg/dL) 17.0 ± 1.07 17.8 ± 0.54 35.7 ± 1.66 a 28.1 ± 1.07 a,b Cr (mg/dL) 0.30 ± 0.02 0.28 ± 0.02 0.51 ± 0.03 a 0.42 ± 0.03 a Urine protein (mg/dL) 11.2 ± 1.14 10.7 ± 0.91 14.8 ± 0.86 a 10.8 ± 0.62 b Urine protein/Cr (mg/mg) 0.28 ± 0.03 0.28 ± 0.02 0.39 ± 0.03 a 0.26 ± 0.01 b CCr (mL/min) 2.13 ± 0.24 2.39 ± 0.19 0.99 ± 0.10 a 1.29 ± 0.07 a,b Daily urine volume (mL) 22.2 ± 1.14 23.9 ± 0.38 18.2 ± 0.78 a 18.5 ± 0.75 a a p < 0.05 compared with the control group; b p < 0.05 compared with the CsA group. CDCA treatment decreased CsA-induced glomerular and tubular damage Histopathological examination of the renal tissues indicated that CsA application led to an increase of glomerular size and enlarged tubule lumens, some of which were filled with cell debris. However, CDCA-treatment was observed to regress glomerular and tubular deformities in CsA rats (Fig. 1 ). The number of glomerular and tubular lesions was calculated to be 1.62 ± 0.18 and 2.25 ± 0.16, respectively, in rats treated with CsA. Treatment with CDCA resulted in a reduction in glomerular (0.75 ± 0.16) and tubular (1.12 ± 0.12) damage scores in CsA rats (p = 0.007; p = 0.001, respectively). CDCA treatment diminished CsA-induced renal oxidative stress and inflammation Renal ROS formation and TBARS levels increased significantly due to CsA application (p = 0.000; p = 0.006, respectively). These levels diminished (p = 0.008, p = 0.008, respectively) due to CDCA treatment in CsA-rats. However, SOD and GSH-PX activities reduced in CsA-rats (p = 0.012 and p = 0.004, respectively) and CDCA treatment returned these reduced enzyme activities to normal control levels in CsA-rats (Table 4 ). Table 4 The effect of chenodeoxycholic acid (CDCA) on renal reactive oxygen species (ROS) and thiobarbituric acid reacting substances (TBARS) levels, superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activities as well as tumor necrosis factor-alpha (TNF-α) level and myeloperoxidase (MPO) activity in cyclosporine (CsA)-applied rats (Mean ± SEM). Control (n = 7) CDCA (n = 7) CsA (n = 8) CDCA + CsA (n = 8) ROS (Rfu/mg protein) 98.7 ± 4.62 96.4 ± 3.71 130.7 ± 4.86 a 108.4 ± 4.99 b TBARS (pmol/mg protein) 623.5 ± 18.3 610.0 ± 26.3 755.8 ± 31.0 a 632.5 ± 23.2 b SOD (U/mg protein) 22.0 ± 1.03 21.2 ± 0.95 17.7 ± 0.61 a 20.9 ± 0.99 GSH-Px (nmol/min/mg protein) 99.1 ± 2.99 87.4 ± 3.14 78.0 ± 3.89 a 90.6 ± 4.89 Kidney TNF-α (pg/mL) 115.6 ± 2.79 118.0 ± 3.60 165.2 ± 3.92a 124.4 ± 12.0b Kidney MPO (nmol/min/mg protein) 38.3 ± 1.37 38.0 ± 1.23 51.6 ± 3.75 a 43.5 ± 3.74 a a p < 0.05 compared with the control group; b p < 0.05 compared with the CsA group. Immunostaining of 4-HNE, a major bioactive marker of lipid peroxidation diminished significantly (p = 0.001) in kidneys of rats in CDCA + CsA (1.50 ± 0.19) group as compared to CsA (3.12 ± 0.23) group (Fig. 2 ). Significant increases were detected in TNF-α levels and MPO activity (p = 0.001; p = 0.011, respectively) in CsA-rats. CDCA treatment diminished TNF-α levels significantly (p = 0.001), but MPO activity did not alter in CsA rats (Table 4 ). CDCA treatment elevated renal FXR and SHP-1 expressions in CsA-rats Renal mRNA expressions of FXR and SHP-1 remained unchanged in CsA rats. However, these expressions were found to be elevated in CsA-rats following the administration of CDCA, when compared to controls (p = 0.005; p = 0.019), and CsA group (p = 0.000; p = 0.001), respectively (Fig. 3 ). CDCA treatment downregulated NOX4 and NOX2 mRNA expressions and upregulated Nrf2 mRNA expression in kidney of CsA-rats NOX4 and NOX2 mRNA expressions were detected to elevate in CsA-rats (p = 0.001; p = 0.001, respectively). They diminished significantly (p = 0.011; p = 0.010, respectively) due to CDCA treatment in CsA rats. However, decreased Nrf2 expression was found in kidneys of CsA-rats as compared to controls (p = 0.049). This expression elevated significantly in CsA + CDCA group as compared to controls (p = 0.038) and CsA group (p = 0.000) (Fig. 3 ). CDCA treatment elevated renal HIF-1α and VEGF mRNA expressions in CsA-rats mRNA expressions of HIF-1α and VEGF mRNA diminished significantly in CsA-rats (p = 0.004; p = 0.002, respectively). CDCA treatment resulted in increases in these expressions in CsA rats (p = 0.001; p = 0.008, respectively) (Fig. 3 ). CDCA treatment diminished serum Ang-II levels and upregulated renal mRNA expressions of AT2R and ACE2 in CsA-rats Administration of CsA resulted in significant increases in Ang-II levels in serum of rats as compared to controls (p = 0.003). However, CDCA treatment diminished serum Ang-II levels in CsA-rats (p = 0.040). Moreover, serum Ang 1–7 levels did not alter significantly in CsA and CDCA + CsA groups (Fig. 4 ). mRNA expression of ACE increased significantly in kidneys of CsA-applied rats as compared to controls (p = 0.008), but ACE2 mRNA expression remained unchanged. Although there were no changes in renal mRNA expression of ACE, mRNA expression of ACE2 were found to be significantly elevated (p = 0.004) due to CDCA treatment in CsA rats. In addition, the ratio of ACE/ACE2 mRNA expressions diminished significantly in CDCA + CsA group as compared to CsA group (p = 0.000). Renal AT1R and AT2R mRNA expressions did not alter in CsA-rats. AT1R mRNA expression remained unchanged, but AT2R mRNA expression was highly elevated (p = 0.001) in CsA + CDCA group as compared to control (p = 0.001) and CsA group (p = 0.001). Furthermore, the ratio of AT1R/AT2R expressions was found to be elevated in CsA-treated rats in comparison to controls (p = 0.021). However, this ratio was observed to diminish in the kidneys of the CsA + CDCA group in comparison to the control (p = 0.001) and the CsA group (p = 0.001) (Fig. 4 ). Discussion Nephrotoxicity remains frequently stated problems associated with CsA. Consequently, ongoing efforts are dedicated to discovering effective methods for reducing or preventing this toxicity. On the other hand, FXR agonists have been reported to improve renal functional and structural changes by ameliorating prooxidant-antioxidant balance, suppressing inflammation and inhibiting renal lipid accumulation, and fibrogenesis in experimental nephrotoxicity (Kim et al. 2023 ; Yang et al. 2024 ). According to this, whether CDCA, an endogenous FXR agonist may be protective in CsA-induced renal toxicity was investigated for the first time. Several investigators have examined the structural and functional changes in CsA nephrotoxicity in experimental animals applied CsA in different doses, durations and ways (Hagar et al. 2006 ; Chia et al. 2013 ; Bekpınar et al. 2018; Tan et al. 2020 ; Wei et al., 2021 ; Nouri et al. 2022; Kalaycı et al. 2023 ). In the current study, CsA reduced body weight and kidney weight, but not kidney index in rats. It also elevated serum BUN, Cr, urinary protein levels and reduced CCr values. CsA nephrotoxicity was also confirmed by presence of renal glomerular and tubular histopathological lesions. These results are consistent with those obtained in rodents administered CsA at similar doses and duration (Chia et al. 2013 ; Bekpınar et al. 2018; Tan et al., 2020 ; Wei et al.2021). CDCA was administered to rats for 15 days in this study. No change was found in the mRNA expressions of FXR and its target gene SHP-1 and these expressions increased in the kidney of CsA-rats due to CDCA treatment. Similarly, CDCA treatment was reported to increase FXR and SHP-1 mRNA expressions in high fructose diet-induced renal damage, and CDCA-induced FXR activation can prevent HFrD-induced renal damage in rats (Hu et al. 2012 ). Our study results indicate that CDCA treatment led to a reduction in serum BUN and urinary protein levels, as well as a decrease in CCr values in CsA-treated rats. Moreover, CDCA resulted in an alleviation in histopathological findings in CsA rats. On the other hand, CsA was detected to increase renal oxidative stress parameters in rodents (Hagar et al. 2006 ; Tan et al. 2020 ; Nouri et al. 2022; Kalaycı et al. 2023 ). Moreover, Nrf2 transcription factor and its target hem oxygenase-1 (HO-1) protein levels (Wei et al. 2021 ; Nouri et al. 2022) and antioxidant enzyme activities/expressions (Hagar et al. 2006 ; Tan et al. 2020 ; Nouri et al. 2022; Kalaycı et al. 2023 ) were found to diminish in kidney tissue in CsA-rats. CDCA administration diminished high levels of ROS and TBARS levels and 4-HNE protein expression, but caused upregulation of Nrf2 mRNA expression, and normalized SOD and GSH-Px activities CsA-rats. CsA was detected to increase renal oxidative stress parameters in rodents (Hagar et al. 2006 ; Tan et al. 2020 ; Nouri et al. 2022; Kalaycı et al. 2023 ). Moreover, Nrf2 transcription factor and its target hem oxygenase-1 (HO-1) protein levels (Wei et al. 2021 ; Nouri et al. 2022) and antioxidant enzyme activities/expressions (Hagar et al. 2006 ; Tan et al. 2020 ; Nouri et al. 2022; Kalaycı et al. 2023 ). were found to diminish in kidney tissue in CsA-rats. However, in our study, CDCA administration diminished high levels of ROS and TBARS levels and 4-HNE protein expression, but caused upregulation of Nrf2 mRNA expression, and normalized SOD and GSH-Px activities CsA-rats. On the other hand, it has been reported that NADPH oxidase 4 (NOX4) is the NOX isoform with the highest expression in the kidney, while NOX1 and NOX2 are expressed at a lower levels and play an important role in CsA-induced oxidative stress (Rajaram et al. 2019 ). Indeed, renal NOX4 and NOX2 mRNA/protein expressions were upregulated in CsA-rodents (Djamali et al. 2016 ; Tan et al. 2020 , Bekpınar et al. 2019; Kalaycı et al. 2023 ). In our study, renal NOX4 and NOX2 expressions were also upregulated in CsA-rats, and these expressions decreased due to CDCA, but did not return to normal levels. Moreover, in our study, renal NF-κB, TNF-α and IL-6 levels, and MPO activity were detected to elevate in CsA rodents as previously reported (El-Kashef et al. 2018 ; Harb et al. 2021 ; Nouri et al. 2022), but CDCA treatment decreased TNF-α level but not MPO activity. All results show that CDCA treatment alleviated renal oxidative stress by decreasing ROS- induced lipid peroxidation and activating Nrf2 and antioxidant enzymes, and reducing inflammation in CsA rats. Renal vasoconstriction and hypoxia are critical factor in CsA nephrotoxicity and oxidative stress and RAS activation promotes hypoxia (Yoon and Yang 2009 ; Hoskova et al. 2017). HIF-1α, a transcription factor, is very important in adaptation to hypoxia. Hypoxia prevent degradation of HIF-1α by inhibiting prolyl hydroxylation. Then, it passes into the nucleus and binds to HIF-1β to form a HIF-1 dimer. HIF-1 dimer initiates the expressions of several genes including VEGF to correct renal blood flow (Liu et al. 2022 ). CsA was reported to enhance HIF-1α degradation by increasing ubiquinitation, and contributes to renal toxicity by disturbing the adaptation to hypoxia (Harb et al. 2021 ). In this study, renal HIF-1α and VEGF protein/gene expressions were detected to decrease in CsA-administered rats as previously reported (Sereno et al. 2014 ; Patel and Thaker 2015 ; Harb et al. 2021 ). Moreover, CDCA treatment caused significant increases in renal HIF-1α and VEGF mRNA expressions. Our results indicate that renal HIF-1α and VEGF mRNA upregulations may exert a role in the protective impact of CDCA in CsA-nephrotoxity. RAS activation has a key role in the harmful impacts of CsA on the kidneys and vessels (Yoon and Yang 2009 , Hoskowa et al. 2017). The levels of renin and Ang-II in serum and kidneys were reported to be increased in CsA-rats (Nishiyama et al. 2003 ; Bekpınar et al. 2019; Hu et al. 2022 ; Kalaycı et al. 2023 ). Increased renal ACE was found, but unchanged AT1R mRNA expression (Bekpinar et al 2019 ). Contrarily, increases in AT1R, but decreases in AT2R mRNA expressions were detected in kidney (Nishiyama et al 2003 ). This increase in AT2R activation was postulated as an adaption to attenuate CsA-nephrotoxicity (Nishiyama et al 2003 ). However, recently, renal and aortic mRNA expressions of ACE and AT1R were detected to increase in CsA-rats [7]. Nonetheless, experimental studies have shown that RAS inhibitors were effective in the amelioration of kidney functions, oxidative stress and inflammation in rodents (Nishiyama et al. 2003 ; Yoon et al. 2009: Hu et al. 2022 ). There is no in vivo study on relationship between RAS and FXR. However, in an in vitro condition, FXR agonists (CDCA and GW4064) did not alter mRNA expressions of angiotensinogen and AT1R, but increased the AT2R mRNA expression in rat vascular endothelial cells (Zhang et al. 2016 ). In our study, CDCA diminished high levels of serum Ang II in CsA-rats. CsA application elevated ACE mRNA expression, but it did not alter AT1R, AT2R and ACE2 mRNA expressions in the kidney. Although CDCA did not affect ACE and AT1R expressions, it upregulated AT2R and ACE2 mRNA expressions. Moreover, the ratios of AT1R/AT2R and ACE/ACE2 mRNA expressions was detected to decrease in CDCA + CsA group as compared to CsA group. These results indicate that CDCA inhibited CsA-induced RAS activation in the kidney by upregulating ACE2 and especially AT2R in rats. Thus, ACE2 and AT2R upregulations induced by CDCA may exert to contribute the protective effect against CsA nephrotoxicity by inhibiting RAS activation. Conclusion In summary, our results show that CDCA treatment was effective against CSA-induced nephrotoxicity. CDCA exerted this protective potential by (a) suppressing ROS formation and lipid peroxidation, (b) reducing mRNA expressions of NOX4 and NOX2, which are effective in ROS formation, (c) increasing the expression of NRf2, and the activities of antioxidant enzymes, (d) decreasing inflammation, (e) upregulating HIF-1 alpha and VEGF mRNA expressions, (f) suppressing the classical axis of RAS by upregulation of AT2R and ACE2 in the kidneys of CsA-applied rats. Declarations Acknowledgement Authors thank to Nergis Demir for her contribution to conduct PCR analyses at Molgen Biotechnology Lab. Author contribution SB and IB conceived and designed research. IB and RK conducted animal model and biochemical experiments. VO and MST performed histopathologic examination and immuno-histochemical tests. SB and MU analyzed data and wrote the manuscript. All authors read and approved the manuscript. Funding This work was supported by Research Funds from Istanbul University to project No: TSA-2019-32065. Conflict of interest The authors have declared that there is no conflict of interest. Data availability All data generated or analyzed during this study are included in this published article. Ethics approval All procedures were carried out in accordance with the protocols of the Bezmialem Vakif University Animal Experiments Local Ethics Committee (Approval number: 2019/14). References Al-Rejaie SS, Abuohashish HM, Alkhamees OA, Aleisa AM, Alroujayee AS (2012) Gender difference following high cholesterol diet induced renal injury and the protective role of rutin and ascorbic acid combination in Wistar albino rats. Lipid Health Dis 11: 41. doi: 10.1186/1476-511X-11-41. 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Cite Share Download PDF Status: Published Journal Publication published 04 Dec, 2024 Read the published version in Journal of Molecular Histology → Version 1 posted Editorial decision: Revision requested 14 Jul, 2024 Reviews received at journal 13 Jul, 2024 Reviews received at journal 10 Jul, 2024 Reviewers agreed at journal 03 Jul, 2024 Reviewers agreed at journal 03 Jul, 2024 Reviewers invited by journal 03 Jul, 2024 Editor assigned by journal 30 Jun, 2024 Submission checks completed at journal 29 Jun, 2024 First submitted to journal 25 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4635970","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":326761352,"identity":"cd244d98-c18e-453f-ac53-86020380a3b6","order_by":0,"name":"Ilknur Bingul","email":"data:image/png;base64,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","orcid":"","institution":"Istanbul University","correspondingAuthor":true,"prefix":"","firstName":"Ilknur","middleName":"","lastName":"Bingul","suffix":""},{"id":326761353,"identity":"9a4cd0a1-3c81-4e67-ae32-b62d382215e3","order_by":1,"name":"Rivaze Kalayci","email":"","orcid":"","institution":"Istanbul University","correspondingAuthor":false,"prefix":"","firstName":"Rivaze","middleName":"","lastName":"Kalayci","suffix":""},{"id":326761354,"identity":"ef7b3067-66ff-4f58-9c16-d8c9bbf4776a","order_by":2,"name":"Merva Soluk Tekkesin","email":"","orcid":"","institution":"Istanbul University","correspondingAuthor":false,"prefix":"","firstName":"Merva","middleName":"Soluk","lastName":"Tekkesin","suffix":""},{"id":326761355,"identity":"c5717dea-3768-41b5-97b8-46578c25ee9b","order_by":3,"name":"Vakur Olgac","email":"","orcid":"","institution":"Istanbul University","correspondingAuthor":false,"prefix":"","firstName":"Vakur","middleName":"","lastName":"Olgac","suffix":""},{"id":326761356,"identity":"6eb14906-3a4e-43b5-a51c-d5266a83c563","order_by":4,"name":"Seldag Bekpinar","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Seldag","middleName":"","lastName":"Bekpinar","suffix":""},{"id":326761357,"identity":"6af97378-9f8c-4bd2-8f50-2016c65645b3","order_by":5,"name":"Mujdat Uysal","email":"","orcid":"","institution":"Istanbul University","correspondingAuthor":false,"prefix":"","firstName":"Mujdat","middleName":"","lastName":"Uysal","suffix":""}],"badges":[],"createdAt":"2024-06-25 11:14:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4635970/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4635970/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10735-024-10308-z","type":"published","date":"2024-12-04T15:57:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60825619,"identity":"c8326b90-95f3-4966-afd4-dee4c03b463c","added_by":"auto","created_at":"2024-07-22 14:03:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":63578435,"visible":true,"origin":"","legend":"\u003cp\u003eIn comparison with control group (A), cyclosporine treatment caused some deformations in glomeruli and tubules as shown with arrows. Cellular adhesions were seen between the glomerular segment and Bowman's capsule (C1). Dilatation of the tubules (C2) and cells spilling into the tubule lumen (CAST formation, C3) were also observed in Cyclosporine group. In the Cyclosporine + CDCA group, the histopathological changes induced by cyclosporine, continued with a slight decrease (D). CDCA-treated rats did not show significant morphological alterations compared with the control group (B). Original magnification, ×400 [Colour online].\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4635970/v1/00c03511b8a60c35bb1ea831.png"},{"id":60825618,"identity":"d34ebb3a-7868-4641-a50d-18c334ef2b1c","added_by":"auto","created_at":"2024-07-22 14:03:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":38395275,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentatives of the 4- hydroxynonenal (4-HNE)-immunostained sections of kidney, a sensitive marker of lipid peroxidation (original magnification, ×400, n =7-8 in each group) showing a profound increase in 4-HNE in mostly tubular epithelial cells of cyclosporine-treated group (C). Whereas fewer positive cells were detected in tubular epithelial cells of CDCA co-treated group (D). Immunostaining in CDCA-treated rats (B) did not show any alteration as compared with those in control (A) [Colour online].\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4635970/v1/2af68ed9bc5eab828559ab46.png"},{"id":60825615,"identity":"9c1f892b-7b5d-47c9-80d5-7f3e032347c2","added_by":"auto","created_at":"2024-07-22 14:03:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":513465,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of chenodeoxycholic acid (CDCA) on mRNA expressions of farnesoid X receptor (FXR), small heterodimer partner (SHP-1), NADPH oxidase 2 (NOX 4), NADPH oxidase 2 (NOX4), nuclear factor erythroid 2-related factor (Nrf2), hypoxia inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) of kidneys of cyclosporine (CsA)-applied rats (Means±SEM). \u003csup\u003ea\u003c/sup\u003ep\u0026lt;0.05 as compared to control; \u003csup\u003eb\u003c/sup\u003ep\u0026lt;0.05 as compared to CsA.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4635970/v1/2b8ce326f61c56996724f11d.png"},{"id":60825616,"identity":"ecc90078-371b-4041-b37b-c3a4c476d410","added_by":"auto","created_at":"2024-07-22 14:03:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":572318,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of chenodeoxycholic acid (CDCA) on the levels of angiotensin II (Ang-II) and angiotensin-(1-7) (Ang 1-7) in serum and mRNA expressions of angiotensin converting enzyme (ACE), angiotensin converting enzyme 2 (ACE2), the ratio of ACE/ACE2, angiotensin 1 receptor (AT1R), angiotensin 1 receptor 2 (AT2R) and the ratio of AT1R/AT2R in kidneys of cyclosporine (CsA)-applied rats (Means±SEM).\u0026nbsp; \u003csup\u003ea\u003c/sup\u003ep\u0026lt;0.05 as compared to control; \u003csup\u003eb\u003c/sup\u003ep\u0026lt;0.05 as compared to CsA\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4635970/v1/c54cd3164a41e3a2b44faffb.png"},{"id":70964629,"identity":"fb186f46-3259-46ee-bb20-8ce1ff3eac05","added_by":"auto","created_at":"2024-12-09 16:12:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":269438640,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4635970/v1/163e9c72-3f76-46f9-9db2-0a7117e3b967.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chenodeoxycholic acid alleviated the cyclosporine-induced nephrotoxicity by decreasing oxidative stress and suppressing renin-angiotensin system through AT2R and ACE2 mRNA upregulation in rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCyclosporine A (CsA), an immunosuppressant agent, commonly used for prevention of organ rejection after transplantation surgeries, is also utilized for therapy of some autoimmune diseases. However, renal and vascular side effects commonly occur by long-term use of CsA. Proposed mechanisms include imbalance between vasoconstrictor and vasodilator substances, enhanced activation of inflammation, oxidative stress and the renin angiotensin system (RAS) (Yoon and Yang \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Hoskowa et al. 2017).\u003c/p\u003e \u003cp\u003eAnimal studies indicated that CsA alters kidney function markers and histopathological findings along with oxidative stress and inflammation (Hagar et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Chia et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tan et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Nouri et al. 2022; Kalaycı et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). CsA caused endothelial dysfunction, disturbed vasodilation and vasoconstriction reactions and enhanced ROS formation in vascular system (8,9). Moreover, CsA was detected to stimulate renal and aortic RAS activation (Hu et al. 2002; Nishiyama et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Bekpinar et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kalaycı et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRAS takes role in blood pressure, water, electrolyte balance in the organism and it consists of two axes as classical and alternative axes. The components of classical RAS are angiotensin converting enzyme (ACE), angiotensin II (Ang-II) and Ang II type-1 receptor (AT1R). The main molecule in RAS is Ang-II and it increases the contractility of vessels by binding to the Ang-II type-1 receptor (AT1R), causing oxidative stress, inflammation, proliferative and fibrotic changes. Other receptor of Ang-II is the AngII type-2 receptor (AT2R). When Ang-II binds to AT2R, it exerts opposite effects (vasodilatory, anti-inflammatory, antiproliferative, antifibrotic). However, the affinity of Ang-II for AT2R is very low [12,13]. On the other hand, the alternative axis consists of ACE2, angiotensin-(1\u0026ndash;7) [Ang-(1\u0026ndash;7)] and Mas receptor (MasR). ACE2 is an enzyme that converts Ang-II to Ang-(1\u0026ndash;7). Ang-(1\u0026ndash;7) shows vasodilator, anti-inflammatory, antiproliferative and antifibrotic effects through MasR (Ma et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Vargas Vargas et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe farnesoid X receptor (FXR) is expressed to a significant extent in the liver, kidney and gastrointestinal tract and activated by bile acids or derivatives of bile acids (BAs). As activated by a ligand, FXR can bind to a DNA response element of multiple genes, including small heterodimer partner (SHP), and activates or represses their transcriptions (Han et al. 2018). FXR was reported to regulate several genes involved bile acid, glucose, lipid and intestinal flora metabolisms, inflammation and oxidative stress stress (Han et al., 2018; Zhang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, FXR agonists were reported to be effective in metabolic and cardiovascular disorders, hepatic steatosis and fibrosis/cirrhosis, acute and chronic renal injuries (Han et al. 2018; Zhang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNatural agonists of FXR are primary [including chenodeoxycholic acid (CDCA) and cholic acid (CA)] and secondary BAs [such as lithocholic acid (LCA) and deoxycholic acid (DCA)]. The order of their potency in activating FXR were detected as CDCA\u0026thinsp;\u0026gt;\u0026thinsp;DCA\u0026thinsp;\u0026gt;\u0026thinsp;LCA\u0026thinsp;\u0026gt;\u0026thinsp;CA.\u003c/p\u003e \u003cp\u003eApart from these natural agonists, synthetic agonists such as obeticholic acid and GW-4064 have been developed. These agonists are more powerful than BAs in activating FXR and they have been extensively utilized in both experimental and clinical studies compared to their natural agonists (Han et al.2018; Zhang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). CDCA is the most potent natural FXR agonist. CDCA was reported to improve glucose tolerance and hepatic fat accumulation induced by high fat diet (Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and dinitrobenzene sulfonic acid (DNBS]-induced ulcerative colitis (Goyal et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) in rats. Moreover, CDCA reduced blood pressure, improved vascular relaxation and contraction responses of mesenteric arteries obtained from spontaneously hypertensive rats (Li et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere are several reports investigating the effect of FXR agonists including CDCA on acute and chronic nephrotoxicity (Kim et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yang J et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). FXR agonists obeticholic acid and GW-4064 were reported to alleviate acute renal injury due to ischemia-reperfusion injury (Gai et al \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and cisplatin (Kim et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Similarly, they alleviated renal damage in mice fed with high fat- high cholesterol diet (Wang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and uninephrectomized obese mice (Gai et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Moreover, INT-767 (FXR/TGR5 agonist) also prevented kidney damage in STZ-induced diabetic mice, db/db mice and obese mice fed on high fat diet (Wang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Similarly, CDCA also inhibited the progression of diabetic nephropathy Kkay mice, an animal model of type 2 diabetes (Takebayashi and Inukai \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and prevented kidney injury induced by high fructose diet in rats (Hu et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the literature, no in vivo study reporting the effects of FXR agonists on CsA-mediated nephrotoxicity is noted but in mouse cut liver slides incubated with CsA, FXR and its target genes were found to be downregulated (Szalowska et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). For this reason, based on the antilipogenic, antioxidant, anti-inflammatory and antifibrotic properties of CDCA, it is conceivable that it may be effective against CsA-induced toxicity. Therefore, the outcomes of CDCA administration on CsA nephrotoxicity was investigated for the first time by determining oxidative stress, inflammation and classical and alternative axes of RAS in rats.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eChemicals\u003c/h2\u003e\n \u003cp\u003eCsA (Sandimmune) from Novartis Pharmaceutical (Switzerland), and CDCA (GP-7061) from Glentham Life Sciences (United Kingdom), and other laboratory chemicals from Sigma-Aldrich (USA) were utilized.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eAnimals and experimental model\u003c/h2\u003e\n \u003cp\u003eMale Wistar rats (6\u0026ndash;8 months old) weighing 300\u0026ndash;320 g were used. They were supplied from Bezmialem University, Experimental Medical Research Institute. They were kept in stainless steel cages (3\u0026ndash;4 rats per cage) under controlled temperature, humidity and 12 h light/dark cycles. No feed and water restrictions were applied to guinea pigs. The protocol for this study was reviewed and approved by the Bezmialem University Animal Care and Use Committee.\u003c/p\u003e\n \u003cp\u003eRats were divided into 4 groups randomly;\u003c/p\u003e\u003cspan\u003e\n \u003cp\u003ea) Control (n\u0026thinsp;=\u0026thinsp;7): Animals received standard rat chow and drinking water without restriction for 15 days.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003eb) CDCA (n\u0026thinsp;=\u0026thinsp;7): CDCA was dissolved freshly in dimethyl sulfoxide (DMSO) and injected to rats intraperitoneally (i.p.) at a dosage of 20 mg/kg daily for 15 days.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003ec) CsA (n\u0026thinsp;=\u0026thinsp;8): Animals received CsA at a dosage 25 mg/kg/day subcutaneously (s.c.) for 12 days.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003ed) CDCA\u0026thinsp;+\u0026thinsp;CsA (n\u0026thinsp;=\u0026thinsp;8): CDCA administration at a dosage 20 mg/kg/day (i.p.) to rats was started 3 days before CsA and continued with CsA at a dosage 25 mg/kg/day (s.c) for a total of 15 days.\u003c/p\u003e\n \u003c/span\u003e\n \u003cp\u003eThe duration and dose of CDCA [18,19] and CsA [4,5] administrations were adjusted according to available studies in literature. DMSO was also applied to control group according to experimental procedure as vehicle. Food and water intake and body weights were detected during the experiment. Rats from each group were transferred separately to metabolic cages to collect urine (24h) samples before last day of experimental protocol, and they were kept at -20\u003csup\u003eO\u003c/sup\u003eC.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eCollecting of samples\u003c/h2\u003e\n \u003cp\u003eIn the last stage of the experiment, rats were anesthetized by ketamine and xylazine HCl injections. Rats were sacrificed to collect whole blood through the cardiac puncture, then the sera were separated by the centrifugation at 1500g for 10 minutes. Kidneys were immediately removed. The cold phosphate buffered saline (PBS; 0.01 M, pH: 7.4) was utilized to prepare 10% homogenates (w/v), and then they were centrifuged at 600xg for 10 min at 4\u0026deg;C and supernatants were utilized for biochemical analyses. The antioxidant enzyme activities were also assessed in the postmitochondrial fraction, which was obtained by centrifuging of the supernatants at 10000xg for 20 min at 4\u003csup\u003eo\u003c/sup\u003eC. All materials were stored at -80\u003csup\u003eo\u003c/sup\u003eC until they were needed for analyses. The left kidney was fixed with formaldehyde for histopathological analyses. Urine samples were centrifuged at 1750xg for 10 min, supernatants were used for analyses.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eKidney function evaluation\u003c/h2\u003e\n \u003cp\u003eThe levels of blood urea nitrogen (BUN) and creatinine (Cr) were estimated with a Cobas-Integra 800 autoanalyser (Roche Diagnostics, Germany). This autoanalyser was also used for the determination of urine protein and urine Cr levels. Glomerul filtration rate was evaluated with Cr clearance test (CCr) and was computed with the formula: CCr (mL/min)\u0026thinsp;=\u0026thinsp;the ratio of urine Cr (mg/dL)x24h urine volume (mL) to serum Cr (mg/dL)x1440.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eThe measurement of serum Ang-II and Ang (1\u0026ndash;7) levels\u003c/h2\u003e\n \u003cp\u003eAng-II (KTE101212, Abbkine, Inc. China) and Ang 1\u0026ndash;7 (KTE101211, Abbkine, Inc. China) determinations were done with enzyme linked-immunosorbent assay (ELISA) kits.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eThe evaluation of renal reactive oxygen species (ROS) and lipid peroxide levels\u003c/h2\u003e\n \u003cp\u003eReactive oxygen species levels (ROS) generation was determined in kidney homogenates incubated with 100 \u0026micro;M 2\u0026prime;,7\u0026prime;-dichlorodihydrofluorescein diacetate (Wang and Joseph \u003cspan class=\"CitationRef\"\u003e1999\u003c/span\u003e). The fluorescence of the reaction was estimated with a Fluoroskan Ascent analyser from Thermo Scientific (USA), with an excitation of 485 nm and emission of 538 nm. Absorbances were given as relative fluorescent unit (RFU/mg protein).\u003c/p\u003e\n \u003cp\u003eRenal thiobarbituric acid reacting substances (TBARS) levels were assessed in rats to evaluate lipid peroxidation (Buege and Aust \u003cspan class=\"CitationRef\"\u003e1978\u003c/span\u003e). Renal homogenates were incubated with Buege-Aust reagent in a boiling water bath. Then, the mixture was cooled and centrifuged at 1000xg for 10 min. Absorbances were recorded at 532 nm and computed with the molar extinction-coefficient value (1.56x10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003eM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and expressed as pmol/mg protein.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eRenal superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activities\u003c/h2\u003e\n \u003cp\u003eSOD activity was measured according to spectrophotometric method described Mylorie et al. (Mylorie et al. \u003cspan class=\"CitationRef\"\u003e1986\u003c/span\u003e). Bovine SOD was used as standard. The difference in absorbances between 0 and 8 min of illumination was detected at 540 nm. Absorbances were calculated as U/mg protein. The cumene hydroperoxide was utilized as a substrate to estimate activity of GSH-PX and absorbances were given as nmol NADPH/min/mg protein (Lawrence and Burk \u003cspan class=\"CitationRef\"\u003e1976\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eDetermination of renal tumor necrosis-alpha (TNF-\u0026alpha;) levels and myeloperoxidase (MPO) activity\u003c/h2\u003e\n \u003cp\u003eTNF-\u0026alpha; levels were measured using an ELISA kit (#MBS 2882073, MyBioSource, Inc., San Diego, USA), and results were expressed as pg/mL. MPO was assayed by determining H\u003csub\u003e2\u003c/sub\u003e0\u003csub\u003e2\u003c/sub\u003e dependent oxidation of o-dianisidine in the kidney homogenates. The molar extinction coefficient value (1.3\u0026times;104 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of oxidised o-dianisidine was utilized for the calculation (Rachmilewitz et al. \u003cspan class=\"CitationRef\"\u003e1993\u003c/span\u003e). Results were given as nmol/min/mg protein.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eDetermination of protein levels\u003c/h2\u003e\n \u003cp\u003eProtein levels were determined spectrophotometrically using bicinchoninic acid (Smith et al. \u003cspan class=\"CitationRef\"\u003e1985\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003emRNA expressions in renal tissue\u003c/h2\u003e\n \u003cp\u003eFXR, SHP-1, NADPH oxidases (NOX4, NOX2), nuclear factor erythroid 2-related factor (Nrf2), hypoxia-inducible factor 1-\u0026alpha; (HIF-1\u0026alpha;), vascular endothelial growth factor (VEGF), AT1R, AT2R, ACE and ACE2 mRNA expressions were determined in renal tissue. For this reason, kidney tissues were homogenized, total RNA was isolated with NucleoSpin RNA Isolation Kit (#740955) from Macherey-Nagel (Germany) and then cDNA synthesis with SCRIPT cDNA Synthesis Kit from Jena Bioscience, GmbH (Jena, Germany) were performed using 5 ng RNA template. Quantitative real time polymerase chain reaction (qRT-PCR) analyses were conducted with the primers pairs of forward and reverse acquired from LGC Biosearch Technologies (Denmark), as detailed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, in a RT-PCR system (Biorad CFX Connect, California, USA). The 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e formula was utilized for computing the mRNAs expressions.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePrimer sequences used for qRT-PCR analysis of target mRNA.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"2\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFXR\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eF: 5\u0026apos;-CATTACAACGCGCTCACCTG-3\u0026apos;;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026apos;- TTCCTTAGCCGGCAATGGTG-3\u0026apos;\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSHP-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF: 5\u0026apos;-AGTCTTTCTGGAGCCTTGAGC-3\u0026apos;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026apos;-CAGGACTTCACACAATGCCC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNOX4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF: 5\u0026prime;-CGTCCACCGTTACCAGACAA-3\u0026prime;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026prime;-TTGGCCTCTGCGTATTCGTT-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNOX2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF: 5\u0026rsquo;-TGGCGATCTCAGCAAAAGGTGG-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026rsquo;-GTACTGTCCCACCTCCATCTTG-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNrf2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF: 5\u0026rsquo;-GTGGATCTGTCAGCTACTCCC-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026rsquo;-CTGGGAATATCCAGGGCAAGC-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHIF-1\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF:5\u0026rsquo;-ATGTACCCTAACAAGCCGGG-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026rsquo;-AAGCACGTCATAGGCGGTTT-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVEGF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF:5\u0026rsquo;-GCACTGGACCCTGGTTTAC-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR:5\u0026rsquo;-GGGTCTCAATTGGACGGCAA-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAT1R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF: 5\u0026apos;-TTCACCCTGCCTCAGGATCT-3\u0026apos;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026apos;-CCAGACCCACCAATCCATCC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAT2R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF:5\u0026apos;-AGAAGGAATCCCTGGCAAGC-3\u0026apos;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026apos;-TAAGGCAATCCCAGCAGACC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF: 5\u0026apos;-CGTCCACCGTTACCAGACAA-3\u0026apos;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026apos;-TTGGCCTCTGCGTATTCGTT-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACE2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF: 5\u0026apos;-GAATGCGACCATCAAGCGTC-3\u0026apos;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026apos;-CAAGCCCAGAGCCTACGATT-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGAPDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF: 5\u0026prime;-CAGGG CTGCCTTCTCTTGTG-3\u0026prime;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026prime;-AACTTGCCGTGGGTAGAG TC-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eHistological analysis\u003c/h2\u003e\n \u003cp\u003eKidney tissue was fixed with 10% buffered formalin solution and embedded in paraffin. Sections were cut at 5 \u0026micro;m and stained with hematoxylin\u0026ndash;eosin (H\u0026amp;E) and then histopathological changes were examined by light microscopy. Renal damage was evaluated and scored conducted in accordance with the protocol outlined by Al-Rejaie et al (\u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eRabbit anti-4-hydroxynonenal (4-HNE) primary antibody (Catalog No. orb 100588; Biorbyt, Cambridgeshire, UK) and kidney sections were incubated, and renal 4-HNE were semiquantified by immunostaining. Additionally, positive control studies were conducted in sections of healthy human kidneys. The presence or absence of brown staining was considered indicative of a positive or negative result for each antibody, respectively. Digital photographs were assessed using the Olympus AnalySIS 5 image analysis program.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eIn this study, \u0026lsquo;Statistical Package for Social Sciences Program\u0026rsquo; (21.0; SPSS Inc., Chicago, IL, USA) were utilized for statistical analysis. The values were given as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Under parametric or non-parametric test conditions, mean values were compared between groups by One-way ANOVA test (post-hoc Tukey\u0026rsquo;s test) or Kruskal-Wallis test (post-hoc Mann Whitney-U test), respectively. A value of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBody and kidney weights changes and kidney index\u003c/h2\u003e \u003cp\u003eSignificant decreases in final body weights were found in CDCA (14.0%; p\u0026thinsp;=\u0026thinsp;0.000), CsA (13.3%; p\u0026thinsp;=\u0026thinsp;0.000) and CDCA\u0026thinsp;+\u0026thinsp;CsA (24.0%; p\u0026thinsp;=\u0026thinsp;0.000) groups as compared to controls. In CDCA\u0026thinsp;+\u0026thinsp;CsA group, final body weights of rats are significantly lower than those of CsA-rats (p\u0026thinsp;=\u0026thinsp;0.000). Kidney weights diminished significantly in CsA and CDCA\u0026thinsp;+\u0026thinsp;CsA groups (p\u0026thinsp;=\u0026thinsp;0.005; p\u0026thinsp;=\u0026thinsp;0.001, respectively), but kidney index values remained unchanged as compared to controls (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe effect of chenodeoxycholic acid (CDCA) on body and kidney weights and indexes in cyclosporine (CsA)-treated rats (Means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCDCA\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCsA\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCDCA\u0026thinsp;+\u0026thinsp;CsA\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInitial body weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e322.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e330.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e331.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e311.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFinal body weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e350.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e301.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e303.7\u0026thinsp;\u0026plusmn;\u0026thinsp;7.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e266.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.77\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGain/15 days (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e27.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-28.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.91\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-28.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-45,4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.38\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKidney weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKidney index (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 compared with the control group; \u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 compared with the CsA group.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCDCA treatment alleviated disturbances in renal function markers in CsA-rats\u003c/h2\u003e \u003cp\u003eSerum BUN (p\u0026thinsp;=\u0026thinsp;0.000), Cr (p\u0026thinsp;=\u0026thinsp;0.000) and urinary protein levels (p\u0026thinsp;=\u0026thinsp;0.037) and the ratio of urinary protein level to creatinine level (p\u0026thinsp;=\u0026thinsp;0.002) elevated, but CCr values decreased significantly (p\u0026thinsp;=\u0026thinsp;0.038) in CsA-applied rats. CDCA treatment decreased serum BUN (p\u0026thinsp;=\u0026thinsp;0.01) and urinary protein levels (p\u0026thinsp;=\u0026thinsp;0.012) and the ratio of urinary protein level to Cr level (p\u0026thinsp;=\u0026thinsp;0.006) and increased CCr values (p\u0026thinsp;=\u0026thinsp;0.036) in CsA-rats. Although serum Cr levels decreased by 18.5%, this change was not statistically significant when comparing the CDCA\u0026thinsp;+\u0026thinsp;CsA group to the CsA group (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe effect of chenodeoxycholic acid (CDCA) on blood urea nitrogen (BUN), serum creatinine (Cr) and urine protein levels, creatinine clearance (CCr)values and daily urine volume in cyclosporine (CsA)-applied rats (Means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCDCA\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCsA\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCDCA\u0026thinsp;+\u0026thinsp;CsA\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBUN (mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e17.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e17.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCr (mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrine protein (mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e11.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e10.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrine protein/Cr (mg/mg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCr (mL/min)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDaily urine volume (mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e22.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e23.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 compared with the control group; \u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 compared with the CsA group.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCDCA treatment decreased CsA-induced glomerular and tubular damage\u003c/h2\u003e \u003cp\u003eHistopathological examination of the renal tissues indicated that CsA application led to an increase of glomerular size and enlarged tubule lumens, some of which were filled with cell debris. However, CDCA-treatment was observed to regress glomerular and tubular deformities in CsA rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe number of glomerular and tubular lesions was calculated to be 1.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 and 2.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16, respectively, in rats treated with CsA. Treatment with CDCA resulted in a reduction in glomerular (0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16) and tubular (1.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12) damage scores in CsA rats (p\u0026thinsp;=\u0026thinsp;0.007; p\u0026thinsp;=\u0026thinsp;0.001, respectively).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCDCA treatment diminished CsA-induced renal oxidative stress and inflammation\u003c/h2\u003e \u003cp\u003eRenal ROS formation and TBARS levels increased significantly due to CsA application (p\u0026thinsp;=\u0026thinsp;0.000; p\u0026thinsp;=\u0026thinsp;0.006, respectively). These levels diminished (p\u0026thinsp;=\u0026thinsp;0.008, p\u0026thinsp;=\u0026thinsp;0.008, respectively) due to CDCA treatment in CsA-rats. However, SOD and GSH-PX activities reduced in CsA-rats (p\u0026thinsp;=\u0026thinsp;0.012 and p\u0026thinsp;=\u0026thinsp;0.004, respectively) and CDCA treatment returned these reduced enzyme activities to normal control levels in CsA-rats (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe effect of chenodeoxycholic acid (CDCA) on renal reactive oxygen species (ROS) and thiobarbituric acid reacting substances (TBARS) levels, superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activities as well as tumor necrosis factor-alpha (TNF-α) level and myeloperoxidase (MPO) activity in cyclosporine (CsA)-applied rats (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCDCA\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCsA\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCDCA\u0026thinsp;+\u0026thinsp;CsA\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eROS (Rfu/mg protein)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e98.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e96.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e130.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.86\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e108.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.99\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTBARS (pmol/mg protein)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e623.5\u0026thinsp;\u0026plusmn;\u0026thinsp;18.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e610.0\u0026thinsp;\u0026plusmn;\u0026thinsp;26.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e755.8\u0026thinsp;\u0026plusmn;\u0026thinsp;31.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e632.5\u0026thinsp;\u0026plusmn;\u0026thinsp;23.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSOD (U/mg protein)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e22.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e21.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGSH-Px (nmol/min/mg protein)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e99.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e87.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e78.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.89\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e90.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKidney TNF-α (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e115.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e118.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e165.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.92a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e124.4\u0026thinsp;\u0026plusmn;\u0026thinsp;12.0b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKidney MPO (nmol/min/mg protein)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e38.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e38.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.75\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e43.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.74\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 compared with the control group; \u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 compared with the CsA group.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eImmunostaining of 4-HNE, a major bioactive marker of lipid peroxidation diminished significantly (p\u0026thinsp;=\u0026thinsp;0.001) in kidneys of rats in CDCA\u0026thinsp;+\u0026thinsp;CsA (1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19) group as compared to CsA (3.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23) group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSignificant increases were detected in TNF-α levels and MPO activity (p\u0026thinsp;=\u0026thinsp;0.001; p\u0026thinsp;=\u0026thinsp;0.011, respectively) in CsA-rats. CDCA treatment diminished TNF-α levels significantly (p\u0026thinsp;=\u0026thinsp;0.001), but MPO activity did not alter in CsA rats (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCDCA treatment elevated renal FXR and SHP-1 expressions in CsA-rats\u003c/h2\u003e \u003cp\u003eRenal mRNA expressions of FXR and SHP-1 remained unchanged in CsA rats. However, these expressions were found to be elevated in CsA-rats following the administration of CDCA, when compared to controls (p\u0026thinsp;=\u0026thinsp;0.005; p\u0026thinsp;=\u0026thinsp;0.019), and CsA group (p\u0026thinsp;=\u0026thinsp;0.000; p\u0026thinsp;=\u0026thinsp;0.001), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCDCA treatment downregulated NOX4 and NOX2 mRNA expressions and upregulated Nrf2 mRNA expression in kidney of CsA-rats\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNOX4 and NOX2 mRNA expressions were detected to elevate in CsA-rats (p\u0026thinsp;=\u0026thinsp;0.001; p\u0026thinsp;=\u0026thinsp;0.001, respectively). They diminished significantly (p\u0026thinsp;=\u0026thinsp;0.011; p\u0026thinsp;=\u0026thinsp;0.010, respectively) due to CDCA treatment in CsA rats.\u003c/p\u003e \u003cp\u003eHowever, decreased Nrf2 expression was found in kidneys of CsA-rats as compared to controls (p\u0026thinsp;=\u0026thinsp;0.049). This expression elevated significantly in CsA\u0026thinsp;+\u0026thinsp;CDCA group as compared to controls (p\u0026thinsp;=\u0026thinsp;0.038) and CsA group (p\u0026thinsp;=\u0026thinsp;0.000) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eCDCA treatment elevated renal HIF-1α and VEGF mRNA expressions in CsA-rats\u003c/h2\u003e \u003cp\u003emRNA expressions of HIF-1α and VEGF mRNA diminished significantly in CsA-rats (p\u0026thinsp;=\u0026thinsp;0.004; p\u0026thinsp;=\u0026thinsp;0.002, respectively). CDCA treatment resulted in increases in these expressions in CsA rats (p\u0026thinsp;=\u0026thinsp;0.001; p\u0026thinsp;=\u0026thinsp;0.008, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCDCA treatment diminished serum Ang-II levels and upregulated renal mRNA expressions of AT2R and ACE2 in CsA-rats\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAdministration of CsA resulted in significant increases in Ang-II levels in serum of rats as compared to controls (p\u0026thinsp;=\u0026thinsp;0.003). However, CDCA treatment diminished serum Ang-II levels in CsA-rats (p\u0026thinsp;=\u0026thinsp;0.040). Moreover, serum Ang 1\u0026ndash;7 levels did not alter significantly in CsA and CDCA\u0026thinsp;+\u0026thinsp;CsA groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003emRNA expression of ACE increased significantly in kidneys of CsA-applied rats as compared to controls (p\u0026thinsp;=\u0026thinsp;0.008), but ACE2 mRNA expression remained unchanged. Although there were no changes in renal mRNA expression of ACE, mRNA expression of ACE2 were found to be significantly elevated (p\u0026thinsp;=\u0026thinsp;0.004) due to CDCA treatment in CsA rats. In addition, the ratio of ACE/ACE2 mRNA expressions diminished significantly in CDCA\u0026thinsp;+\u0026thinsp;CsA group as compared to CsA group (p\u0026thinsp;=\u0026thinsp;0.000).\u003c/p\u003e \u003cp\u003eRenal AT1R and AT2R mRNA expressions did not alter in CsA-rats. AT1R mRNA expression remained unchanged, but AT2R mRNA expression was highly elevated (p\u0026thinsp;=\u0026thinsp;0.001) in CsA\u0026thinsp;+\u0026thinsp;CDCA group as compared to control (p\u0026thinsp;=\u0026thinsp;0.001) and CsA group (p\u0026thinsp;=\u0026thinsp;0.001). Furthermore, the ratio of AT1R/AT2R expressions was found to be elevated in CsA-treated rats in comparison to controls (p\u0026thinsp;=\u0026thinsp;0.021). However, this ratio was observed to diminish in the kidneys of the CsA\u0026thinsp;+\u0026thinsp;CDCA group in comparison to the control (p\u0026thinsp;=\u0026thinsp;0.001) and the CsA group (p\u0026thinsp;=\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eNephrotoxicity remains frequently stated problems associated with CsA. Consequently, ongoing efforts are dedicated to discovering effective methods for reducing or preventing this toxicity. On the other hand, FXR agonists have been reported to improve renal functional and structural changes by ameliorating prooxidant-antioxidant balance, suppressing inflammation and inhibiting renal lipid accumulation, and fibrogenesis in experimental nephrotoxicity (Kim et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). According to this, whether CDCA, an endogenous FXR agonist may be protective in CsA-induced renal toxicity was investigated for the first time.\u003c/p\u003e \u003cp\u003eSeveral investigators have examined the structural and functional changes in CsA nephrotoxicity in experimental animals applied CsA in different doses, durations and ways (Hagar et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Chia et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Bekpınar et al. 2018; Tan et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wei et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Nouri et al. 2022; Kalaycı et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In the current study, CsA reduced body weight and kidney weight, but not kidney index in rats. It also elevated serum BUN, Cr, urinary protein levels and reduced CCr values. CsA nephrotoxicity was also confirmed by presence of renal glomerular and tubular histopathological lesions. These results are consistent with those obtained in rodents administered CsA at similar doses and duration (Chia et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Bekpınar et al. 2018; Tan et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wei et al.2021).\u003c/p\u003e \u003cp\u003eCDCA was administered to rats for 15 days in this study. No change was found in the mRNA expressions of FXR and its target gene SHP-1 and these expressions increased in the kidney of CsA-rats due to CDCA treatment. Similarly, CDCA treatment was reported to increase FXR and SHP-1 mRNA expressions in high fructose diet-induced renal damage, and CDCA-induced FXR activation can prevent HFrD-induced renal damage in rats (Hu et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Our study results indicate that CDCA treatment led to a reduction in serum BUN and urinary protein levels, as well as a decrease in CCr values in CsA-treated rats. Moreover, CDCA resulted in an alleviation in histopathological findings in CsA rats.\u003c/p\u003e \u003cp\u003eOn the other hand, CsA was detected to increase renal oxidative stress parameters in rodents (Hagar et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Tan et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Nouri et al. 2022; Kalaycı et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, Nrf2 transcription factor and its target hem oxygenase-1 (HO-1) protein levels (Wei et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Nouri et al. 2022) and antioxidant enzyme activities/expressions (Hagar et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Tan et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Nouri et al. 2022; Kalaycı et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) were found to diminish in kidney tissue in CsA-rats. CDCA administration diminished high levels of ROS and TBARS levels and 4-HNE protein expression, but caused upregulation of Nrf2 mRNA expression, and normalized SOD and GSH-Px activities CsA-rats.\u003c/p\u003e \u003cp\u003eCsA was detected to increase renal oxidative stress parameters in rodents (Hagar et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Tan et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Nouri et al. 2022; Kalaycı et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, Nrf2 transcription factor and its target hem oxygenase-1 (HO-1) protein levels (Wei et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Nouri et al. 2022) and antioxidant enzyme activities/expressions (Hagar et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Tan et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Nouri et al. 2022; Kalaycı et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). were found to diminish in kidney tissue in CsA-rats. However, in our study, CDCA administration diminished high levels of ROS and TBARS levels and 4-HNE protein expression, but caused upregulation of Nrf2 mRNA expression, and normalized SOD and GSH-Px activities CsA-rats. On the other hand, it has been reported that NADPH oxidase 4 (NOX4) is the NOX isoform with the highest expression in the kidney, while NOX1 and NOX2 are expressed at a lower levels and play an important role in CsA-induced oxidative stress (Rajaram et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Indeed, renal NOX4 and NOX2 mRNA/protein expressions were upregulated in CsA-rodents (Djamali et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Tan et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Bekpınar et al. 2019; Kalaycı et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In our study, renal NOX4 and NOX2 expressions were also upregulated in CsA-rats, and these expressions decreased due to CDCA, but did not return to normal levels.\u003c/p\u003e \u003cp\u003eMoreover, in our study, renal NF-κB, TNF-α and IL-6 levels, and MPO activity were detected to elevate in CsA rodents as previously reported (El-Kashef et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Harb et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Nouri et al. 2022), but CDCA treatment decreased TNF-α level but not MPO activity. All results show that CDCA treatment alleviated renal oxidative stress by decreasing ROS- induced lipid peroxidation and activating Nrf2 and antioxidant enzymes, and reducing inflammation in CsA rats.\u003c/p\u003e \u003cp\u003eRenal vasoconstriction and hypoxia are critical factor in CsA nephrotoxicity and oxidative stress and RAS activation promotes hypoxia (Yoon and Yang \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Hoskova et al. 2017). HIF-1α, a transcription factor, is very important in adaptation to hypoxia. Hypoxia prevent degradation of HIF-1α by inhibiting prolyl hydroxylation. Then, it passes into the nucleus and binds to HIF-1β to form a HIF-1 dimer. HIF-1 dimer initiates the expressions of several genes including VEGF to correct renal blood flow (Liu et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). CsA was reported to enhance HIF-1α degradation by increasing ubiquinitation, and contributes to renal toxicity by disturbing the adaptation to hypoxia (Harb et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this study, renal HIF-1α and VEGF protein/gene expressions were detected to decrease in CsA-administered rats as previously reported (Sereno et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Patel and Thaker \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Harb et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, CDCA treatment caused significant increases in renal HIF-1α and VEGF mRNA expressions. Our results indicate that renal HIF-1α and VEGF mRNA upregulations may exert a role in the protective impact of CDCA in CsA-nephrotoxity.\u003c/p\u003e \u003cp\u003eRAS activation has a key role in the harmful impacts of CsA on the kidneys and vessels (Yoon and Yang \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Hoskowa et al. 2017). The levels of renin and Ang-II in serum and kidneys were reported to be increased in CsA-rats (Nishiyama et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Bekpınar et al. 2019; Hu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kalaycı et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Increased renal ACE was found, but unchanged AT1R mRNA expression (Bekpinar et al \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Contrarily, increases in AT1R, but decreases in AT2R mRNA expressions were detected in kidney (Nishiyama et al \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). This increase in AT2R activation was postulated as an adaption to attenuate CsA-nephrotoxicity (Nishiyama et al \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). However, recently, renal and aortic mRNA expressions of ACE and AT1R were detected to increase in CsA-rats [7]. Nonetheless, experimental studies have shown that RAS inhibitors were effective in the amelioration of kidney functions, oxidative stress and inflammation in rodents (Nishiyama et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Yoon et al. 2009: Hu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere is no in vivo study on relationship between RAS and FXR. However, in an in vitro condition, FXR agonists (CDCA and GW4064) did not alter mRNA expressions of angiotensinogen and AT1R, but increased the AT2R mRNA expression in rat vascular endothelial cells (Zhang et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In our study, CDCA diminished high levels of serum Ang II in CsA-rats. CsA application elevated ACE mRNA expression, but it did not alter AT1R, AT2R and ACE2 mRNA expressions in the kidney. Although CDCA did not affect ACE and AT1R expressions, it upregulated AT2R and ACE2 mRNA expressions. Moreover, the ratios of AT1R/AT2R and ACE/ACE2 mRNA expressions was detected to decrease in CDCA\u0026thinsp;+\u0026thinsp;CsA group as compared to CsA group. These results indicate that CDCA inhibited CsA-induced RAS activation in the kidney by upregulating ACE2 and especially AT2R in rats. Thus, ACE2 and AT2R upregulations induced by CDCA may exert to contribute the protective effect against CsA nephrotoxicity by inhibiting RAS activation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our results show that CDCA treatment was effective against CSA-induced nephrotoxicity. CDCA exerted this protective potential by (a) suppressing ROS formation and lipid peroxidation, (b) reducing mRNA expressions of NOX4 and NOX2, which are effective in ROS formation, (c) increasing the expression of NRf2, and the activities of antioxidant enzymes, (d) decreasing inflammation, (e) upregulating HIF-1 alpha and VEGF mRNA expressions, (f) suppressing the classical axis of RAS by upregulation of AT2R and ACE2 in the kidneys of CsA-applied rats.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u0026nbsp;\u003c/strong\u003eAuthors thank to Nergis Demir for her contribution to conduct PCR analyses at Molgen Biotechnology Lab.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSB and IB conceived and designed research. IB and RK conducted animal model and biochemical experiments. VO and MST performed histopathologic examination and immuno-histochemical tests. SB and MU analyzed data and wrote the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Research Funds from Istanbul University to project No: TSA-2019-32065.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eThe authors have declared that there is no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003eAll procedures were carried out in accordance with the protocols of the Bezmialem Vakif University Animal Experiments Local Ethics Committee (Approval number: 2019/14).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAl-Rejaie SS, Abuohashish HM, Alkhamees OA, Aleisa AM, Alroujayee AS (2012) Gender difference following high cholesterol diet induced renal injury and the protective role of rutin and ascorbic acid combination in Wistar albino rats. 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Clin Exp Pharmacol Physiol 43: 327-34. doi: 10.1111/1440-1681.12535.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-histology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hijo","sideBox":"Learn more about [Journal of Molecular Histology](https://www.springer.com/journal/10735)","snPcode":"10735","submissionUrl":"https://submission.springernature.com/new-submission/10735/3","title":"Journal of Molecular Histology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Chenodeoxycholic acid, Cyclosporine A, Oxidative stress, Nephrotoxicity, Renin angiotensin system ","lastPublishedDoi":"10.21203/rs.3.rs-4635970/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4635970/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOxidative stress, inflammation and renin-angiotensin system (RAS) activation play an important role in the nephrotoxicity which is caused by the long-term use of the immunosuppressive drug cyclosporine (CsA). This study investigates whether chenodeoxycholic acid (CDCA), an endogenous farnesoid X receptor (FXR) agonist with antioxidant and anti-inflammatory effects, modulates CsA nephrotoxicity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCsA (25 mg/kg/day; s.c.) was administered to rats for 12 days. CDCA (20 mg/kg/day; i.p.) injection was started 3 days before CsA and continued for 15 days. CDCA improved renal damage and function in CsA-administered rats. Renal function markers in serum, renal histology, oxidative stress, inflammation and RAS components were determined in kidney.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCDCA reduced CsA-induced renal increases in NADPH oxidases 4 and NADPH oxidases 2 mRNA expressions, oxidative stress and inflammation. CDCA elevated renal FXR, small heterodimer partner-1, hypoxia-inducible factor and vascular endothelial growth factor and nuclear factor erythroid 2-related factor mRNA expressions in CsA rats. It prevents renin angiotensin system activation by reducing angiotensin II (Ang-II) levels in serum and upregulating renal mRNA expressions of Ang II type-II receptor (AT2R) and angiotensin converting enzyme 2 (ACE2), but not AT1R and ACE in CsA rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur results indicate that CDCA may be a protective agent against CsA-nephrotoxicity by decreasing inflammation, oxidative stress and RAS activation via AT2R and ACE2 upregulations.\u003c/p\u003e","manuscriptTitle":"Chenodeoxycholic acid alleviated the cyclosporine-induced nephrotoxicity by decreasing oxidative stress and suppressing renin-angiotensin system through AT2R and ACE2 mRNA upregulation in rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-22 14:03:51","doi":"10.21203/rs.3.rs-4635970/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-14T14:58:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-13T18:12:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-11T03:45:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"52208421753002947024400573258635901638","date":"2024-07-04T00:58:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36558447245835089257995599933463232011","date":"2024-07-03T13:27:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-03T12:41:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-30T19:36:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-29T07:48:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Molecular Histology","date":"2024-06-25T11:11:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-histology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hijo","sideBox":"Learn more about [Journal of Molecular Histology](https://www.springer.com/journal/10735)","snPcode":"10735","submissionUrl":"https://submission.springernature.com/new-submission/10735/3","title":"Journal of Molecular Histology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c9590dd9-0859-4028-bfee-abb5d9fcc839","owner":[],"postedDate":"July 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-09T15:59:17+00:00","versionOfRecord":{"articleIdentity":"rs-4635970","link":"https://doi.org/10.1007/s10735-024-10308-z","journal":{"identity":"journal-of-molecular-histology","isVorOnly":false,"title":"Journal of Molecular Histology"},"publishedOn":"2024-12-04 15:57:00","publishedOnDateReadable":"December 4th, 2024"},"versionCreatedAt":"2024-07-22 14:03:51","video":"","vorDoi":"10.1007/s10735-024-10308-z","vorDoiUrl":"https://doi.org/10.1007/s10735-024-10308-z","workflowStages":[]},"version":"v1","identity":"rs-4635970","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4635970","identity":"rs-4635970","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}