Glycerol-induced rhabdomyolysis in rats establishes adjacent alikeness to rhabdomyolysis in humans, with respect to histopathological lineaments and cytokine profile (Song, Kim et al. 2020).
This study showed that Gly-induced renal injury was proved by elevation in levels of serum and urine creatinine, urine albumin, urine T. protein and BUN, in addition to irregular histological profile: acute tubular necrosis, brush border loss, tubular dilation with cast formation. These abnormalities might be ascribed to initiation of the inflammation cascade and generation of ROS, leading to the disruption and degeneration of structural integrity in kidney cells, and consequently, renal damage, as mentioned with numerous earlier studies (Gu, Yang et al. 2014, Li, Xu et al. 2019). Moreover, in this research, Gly caused a remarkable increase in mortality, and RSI, which is expected to be a consequence of the above mentioned proteinuria, albuminuria and renal damage.
Meanwhile, the studied drugs in order of potency: DATS 20 mg, followed by DATS 10 mg, then lastly, sildenafil maintained the overall body health. They produced significant alleviation in Gly-induced renal injury, histopathological manifestations and RSI, as verified by the significant decrease in all elevated renal function tests, retention of normal histological architecture and improvement of survival.
Furthermore, intramuscular injection of glycerol augmented levels of CK and its isoenzyme CK-MB, leading to general skeletal muscle injury and release of myoglobin into blood that in turn caused AKI (Holt and Moore 2000). Treatment with sildenafil and DATS (20 mg/kg) achieved improvement in Gly-induced acute muscle injury, while the lower dose of DATS (10 mg/kg) was insufficient to alleviate muscle injury. This study indicated for the first time that sildenafil and DATS could be muscle protective drugs, through reversal of the elevated serum levels of total CK and CK-MB in rhabdomyolysis model.
Many mechanisms contribute to glycerol-induced AKI, including: oxidative stress, renal vasoconstriction, inflammation and intra-tubular cast formation. Oxidative stress showed a serious role in the development of RIAKI, as it leads to the damage of renal tubular cells, which subsequently prompts the liberation of cytokines and chemokines, and triggers leukocyte stimulation (Koupenova and Ravid 2013)
In the current study, glycerol-induced oxidative stress was expressed by the elevated renal MDA and NO levels and the decreased renal antioxidant enzymes: SOD and GSH, in addition to the overall renal TAC. The results showed that DATS (20 mg/kg) was able to reverse all Gly-induced oxidant/antioxidant disturbances, therefore owns a remarkable antioxidant activity, which signifies its potential to prevent oxidative and nitrosative damage, while the lower dose of DATS (10 mg/kg) had a lesser pronounced antioxidant effect. These outcomes are in convenience with a previous study, which documented that DATS had a protective effect against oxidative stress (Prabu and Sumedha 2014), in addition to another study, which reported that garlic polysulfide decreased ROS generation and alleviated neutrophil infiltration in the kidney (Ko et al., 2017; Miltonprabu et al., 2017).
The ability of Sildenafil to improve glycerol-induced elevation in free radicals is confirmed in our study via significant elevation of antioxidants, and reduction of lipid peroxidation to fight free radicals. This was reliable with the outcomes of previous studies, which confirmed its antioxidant effects and presented that Sild decreased malondialdehyde in several experimentally-induced kidney injuries in rats (Cadirci, Halici et al. 2011, Küçük, Yucel et al. 2012, Morsy, Ibrahim et al. 2014). Previous reports denoted that Sild was able to increase GSH (Beheshtian, Salmasi et al. 2008), superoxide dismutase (Sanders 2020) and TAC (Iordache, Docea et al. 2020) activities. Meanwhile, Sild had no remarked effect on NO level, as it is well known to increase its constitutive levels in several tissues, without producing nitrosative damage. Consequently, sildenafil (5 mg/kg) can be considered an effective drug in reducing renal oxidative stress, but to a lesser extent than DATS (20 mg/kg), as presented in this work.
AKI is linked with augmented liberation of cytokines, mainly, TNF-α, IL-1β, and IL-6 that can recruit oxidative flow. TNF-α can excite inflammatory consequences by triggering NF-ĸB. NF-κB shows a critical role in organizing a lot of genes involved in inflammation (Papaconstantinou, Zeglinas et al. 2014), including COX-2. Increased NF-κB expression, along with increased TNF-α, IL-1β, IL-6 and COX-2 expressions were previously confirmed to be directly involved in renal injury (McSweeney, Gadanec et al. 2021).
In this study, glycerol caused elevation in renal levels of the pro-inflammatory cytokines: TNF-α, IL-1β, and IL-6, in addition to COX-2. This was confirmed in previous reports, which stated that levels of TNF-α, IL-1β, IL-6, and COX-2 were elevated in experimentally induced rhabdomyolysis (Geng, Zhang et al. 2014, Homsi, Andreazzi et al. 2015, Ahn, Nam et al. 2016).
Meanwhile, DATS, in a dose dependent manner, significantly diminished the production of all inflammatory cytokines. The anti-inflammatory effect of DATS was previously reported in several diseases such as cancer (Jurkowska, Wróbel et al. 2017) and ulcerative colitis (Bai, Ouyang et al. 2005), which is in accordance with our results. Sildenafil produced a remarkable decrease in the production of TNF-α and COX-2, and no variation was observed in the production of other cytokines. This may be due to utility of a single low dose of sildenafil (5 mg/kg).
It is well known that there is a counterbalance between NRF-2 and NF-κB pathways. NF-κB could directly suppress NRF-2 at the transcriptional level (Wardyn, Ponsford et al. 2015). Otherwise, triggering of NRF-2/HO-1 antioxidant signaling weakens inflammatory signaling paths and NF-κB intermediated pro-inflammatory reactions (Saber, Khalil et al. 2019). Moreover, it has been recognized that NRF-2 activators were created to inhibit IκK/IκB phosphorylation and consequently inhibited NF-κB p65 nuclear translocation, thus, deactivating NF-κB signaling (Dharmani, Leung et al. 2011).
In the present experimental study, treatment with sildenafil and DATS, in a dose dependent manner, achieved improvement in rhabdomyolysis-induced disruption in the balance between NRF-2 and NF-κB, by decreasing the expression of NF-κB and increasing the expression of NRF-2 and HO-1 in RIAKI rats. These consequences suggest that DATS efficiently diminishes inflammation via inhibition of NF-κB signal transduction pathway, which was stated in previous reports as well (Pan, Lin et al. 2016, Yang, Tang et al. 2019). Other studies supported our results and confirmed that DATS activates NRF-2/HO-1 pathway (Kim, Lee et al. 2014, Silva-Islas, Chánez-Cárdenas et al. 2019). Therefore, DATS may be considered as a NF-κB inhibitor, and a NRF-2 activator, possibly by its anti-inflammatory effect, as well as its antioxidant effect. In addition, the resulted modulation of these pathways, proved the antioxidant and anti-inflammatory effects of sildenafil, which was confirmed in previous reports (Kniotek and Boguska 2017, Maziero Alves, Aires et al. 2021).
The augmented apoptosis level in rhabdomyolysis, leads to acute kidney injury, and the significant raise in the level of apoptotic Bax is well known to be correlated to the anti-apoptotic BCL-2 protein (Wang, Zhang et al. 2011). In this study, sildenafil and DATS, in a dose-dependent mode, decreased the ratio of Bax/BCL-2 as noticed by immunolabeling. This is coincided with a former study, which confirmed that DATS protected against apoptosis during myocardial ischemia-reperfusion injury in diabetic rats (Yu, Li et al. 2017).