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.