One major adverse effect that prevents CTX from being used in the treatment of cancer is hepatorenal toxicity. It has been shown that CTX can cause oxidative stress-related damage to the liver and kidneys (Mahmoud et al., 2017; Lim et al., 2017). Despite the fact that numerous studies have focused their attention on CTX hepatorenal toxicity, there are still very few effective treatment options available. Here, we looked at how well AD-MSCs Exo protected rats' hepatorenal toxicity caused by CTX. Our findings demonstrated that by upregulating Nrf-2/HO-1, downregulating NF-κB/TLR-4 and Bax/Bcl-2 signaling pathways, and suppressing oxido-inflammatory stress and apoptotic end points in CTX-challenged rats, AD-MSCs Exo may mitigate liver and kidney injury.
In the current investigation, liver transaminases (ALT and AST) activity in serum was significantly elevated in rats treated with CTX; significant indicators for assessing liver damage because their blood leakage indicates the degree of liver damage (Yogalakshmi et al., 2010) where the increased lipid peroxidation caused by CTX changes the fluidity and integrity of the membrane, which in turn disrupts the permeability of the membrane (Catalá and Díaz, 2016) and thereby causes them to leak into the bloodstream. Althunibat et al. (2023) shown that in the rat model of CTX-induced hepatotoxicity, blood levels of ALT and AST were elevated. Additionally, hepatic MDH and GLDH levels were significantly higher in the CTX group compared to the control. These findings are similar to those of Schomaker et al. (2013), who found that acetaminophen toxicity in the liver was associated with higher levels of GLDH and MDH. These elevations may result from hepatic induction of these enzymes in response to specific medications (Shimizu et al., 1997) (like CTX in this study) and glucocorticoids in other studies (Timmerman et al., 2003).
CTX administration also results in considerable elevation in the levels of serum urea and creatinine compared to control group. Similar findings were reported by Alaqeel and Al-Hariri (2023), who found that rats treated with CTX, had elevated serum urea and creatinine levels. They are only seen in considerable quantities in the blood, kidneys, and proximal-distal tubules following renal membrane damage and ischemia (Mori et al., 2005). Therefore, elevated release of these indicators into the bloodstream suggests renal injury resulting from CTX.
In the current investigation, nephrotoxicity resulted in the elevation of KIM-1 protein in kidney tissues. According to results from Ijaz et al. (2022), the renal KIM-1 level increased in the rat model of CTX-induced nephrotoxicity. The hypothesis illustrated the cause of high renal KIM-1 level was attributed to extracellular regulated kinase ½ (ERK½) and signal transducer and activator of transcription 3 (STAT3) phosphorylated pathway where STAT3 bounded to KIM-1 promotor and raised its expression at both mRNA and protein level (Moresco et al., 2018). It was illustrated that CTX was more efficient in upregulation of STAT3 phosphorylation (Noori et al., 2020) and therefore raise renal KIM-1 protein levels through STAT3 binding to KIM-1 promotor.
Furthermore, compared to the control group, the CTX group had an elevated level of renal clusterin protein. The overexpression of the clusterin protein indicates the presence of renal damage and serves as a possible indicator of nephrotoxicity (Girton et al., 2002). Previous research revealed a connection between the TGF-β signaling system and clusterin expression. TGF-β1 activates protein kinase C and AP-1 transcriptor protein, which in turn causes the expression of clusterin (Jin and Howe, 1997). CTX induces TGF-β1 (Iqubal et al., 2023) and thereby caused a rise in renal clusterin protein levels.
According to the current findings, liver and kidney tissues treated with CTX showed a substantial increase in MDA protein concentration and iNOS protein expression when compared to the control. These findings are similar to those of Alaqeel and Al-Hariri (2023), where CTX significantly raised the level of MDA protein and iNOS antibody expression in the renal tissues relative to the control. The active toxic metabolites of CTX, phosphoramide and acrolein, are probably responsible for its anti-malignant effects. Phosphoramide is responsible for CTX's mutagenic effects. On the other hand, acrolein hinders the cellular antioxidant defense system, resulting in highly reactive oxygen species (ROS) formation which interacts with amino acids of the body; which in turn causes morphological and physiological alterations (Caglayan et al., 2018). Additionally, hepatocyte-cytochrome P450 mixed function oxidase enzymes oxidize CTX multiple times in the hepatic tissues to generate oxidative agents like acrolein, which contributes to the overproduction of free radicals like ROS and NO (Althunibat et al., 2023). Lipid peroxidation produces MDA as an end product, and MDA level elevated due to oxidative stress (Mahipal and Pawar, 2017) resulted from CTX. Additionally, NF-κB activated by CTX (Lan et al., 2022) causes iNOS synthesis (Yang et al., 2015) and this led to increased iNOS immunoreactivity.
The findings demonstrated that, in comparison to control, CTX downregulated the Nrf-2/HO-1 signaling pathway in the liver and kidneys. This matches previous research by Mahmoud et al. (2017) and Althunibat et al. (2023) that demonstrated decreased expression of HO-1 and Nrf-2 in the liver in a rat model of CTX hepatotoxicity. Nuclear factor erythroid 2-related factor 2 (Nrf-2) stimulates the production of many antioxidant enzymes in response to reactive oxygen species (ROS), hence suppressing oxidative stress (Satta, et al., 2017). Under normal circumstances, Kelch-like ECH-associated protein 1 (Keap1), a sensor protein towards electrophiles and ROS, sequesters Nrf2 in the cytoplasm. A moderate degree of oxidative stress causes Nrf2 to be released and enter the nucleus, where it attaches to the DNA promoter region's antioxidant response element (ARE) and initiates heme oxygenase (HO)-1 transcription (Satta, et al., 2017). It is believed that the transcription regulator Nrf-2 triggers expression of HO-1, which is among the body's most significant antioxidant systems (Lan et al., 2022). Here, CTX suppressed Nrf-2 signaling as illustrated by Nrf-2 downregulation, and HO-1 gene expression. Despite ROS represent the signal that induces Nrf-2 to dissociate from Keap1 and trigger the antioxidant genes transcription; it inhibited Nrf-2 signaling after injection of CTX. The declined Nrf-2/HO-1 pathway might be due to sustained surplus ROS levels which have been reported to inhibit Nrf-2 in the liver (Abd El-Twab et al., 2019), and the kidney (Mahmoud et al., 2018).
According to the current approach, rats given CTX showed a considerable increase in the levels of TNF-α in their liver and kidney, as well as a high degree of positive expression for the COX-2 antibody in the liver and kidney. It is also upregulated NF-κB/TLR-4 signaling pathway. These results are consistent with those of Lan et al. (2022), who demonstrated that CTX enhanced TNF-α protein levels and raised the expression levels of TLR-4, MyD88, and NF-κB p65 genes in thymus and spleen tissues. NF-κB is a transcription factor that regulates the immune response and many inflammatory illnesses in various tissues. It is essential for the activation of pro-inflammatory cytokines like COX-2 and TNF-α (Semis et al., 2021). Oxidative stress in CTX-treated tissues activates NF-κB, which leads to the generation of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6, which in turn causes tissue damage (Caglayan et al., 2018). Additionally, Nrf-2 inhibits the inflammatory response mediated by NF-κB by reducing the activation of NF-κB triggered by oxidative stress, blocking the proteasomal breakdown of IκB-α, and subsequently blocking the nuclear translocation of NF-κB (Saha et al., 2020). Nevertheless, downregulation of Nrf-2 produced from CTX increased severity of CTX-induced toxicity via NF-κB upregulation. These results showed that Nrf-2 plays a critical role in avoiding drug toxicity, mostly by enhancing the inflammatory response within cells through the NF-κB/TLR-4 signaling pathway.
It was demonstrated in this work that CTX-induced apoptosis. In the liver and kidney tissue of the CTX group, we showed that, in comparison to the control, there was an elevation of the pro-apoptotic marker Bax and a decrease in the expression of the anti-apoptotic Bcl-2. Consistent with our findings, earlier research demonstrated that CTX could cause kidney tissue to undergo apoptosis by upregulating the expression of apoptotic markers such as caspase-3 and Bax (Caglayan et al., 2018). Furthermore, Asiri (2010) reported that in cardiac tissues, CTX dramatically lowers the expression of Bcl-2 and enhances the mRNA expression of P53 and Bax. CTX-induced generation of ROS and therefore increase NF-κB activation, which in turn causes the production of pro-inflammatory mediators. This results in a concerted expression of different pro-apoptotic proteins, such caspases and Bax, or anti-apoptotic proteins, like Bcl-2 (Ullrich et al., 2022). Overproduction of ROS promotes the dissipation of the mitochondrial membrane potential, which in turn allows cytochrome c to be released into the cytosol. Apoptosis activating factor-1 (Apaf-1) and procaspase-9 combine with cytochrome c to form what is known as an apoptosome, this causes caspase-9 to become auto-activated, ultimately resulting in DNA breakage, cleavage of cellular proteins, and cell death through apoptosis via activation of the executioner caspase-3 (Circu and Aw, 2010), all of those are crucial mediators of the intrinsic pathway of pro- and anti-apoptotic signals (Bax and Bcl-2) (Radhiga et al., 2012).
Hepatonephrototoxic effects of CTX were further ascertained by the assessment of histological alterations of the liver and kidneys tissue. Histological examination of the liver tissue of rats given CTX treatment revealed portal fibrosis, hepatic sinusoid dilatation, and engorgement of blood vessels in addition to a substantial infiltration of mononuclear inflammatory cells. Furthermore, histological examination of the kidney tissue of rats given CTX revealed nuclear pyknosis, atrophy of the renal glomeruli, and minor vacuolar degeneration in the epithelial lining of some renal tubules accompanied with moderate renal blood vessel congestion. According to Althunibat et al. (2023), mice administered CTX exhibited pronounced centrilobular hepatic necrosis linked to hepatic vacuolation. Studies by Ijaz et al. (2022) demonstrated that renal tissue inter-tubular vessels, tubular dilatation, glomerular hyperemia, and tubule epithelium underwent degenerative alterations as a result of CTX treatment. These pathological alterations could be linked to CTX's capacity to weaken the antioxidant defense system and produce free radicals. According to several reports, the injection of CTX can directly harm the kidney, resulting in glomerulus degeneration, necrosis in the proximal convoluted tubule, distal tubules, pyknosis, etc (El-shabrawy et al., 2020). In addition to oxidative stress, nephrotoxicity is also significantly influenced by elevated pro-inflammatory cytokines, apoptotic, and fibrotic proteins synthesis (El-shabrawy et al., 2020).
In contrast, the injured liver and kidney tissues showed improved histological characteristics, reduced inflammatory response, decreased apoptosis, and increased antioxidant capacity in both AD-MSCs and AD-MSCs-Exo. These effects may account for the hepatorenal protective properties of both AD-MSCs as well as AD-MSCs-Exo upon CTX-induced tissue damage. These results are consistent with earlier research by El Araby et al. (2022), who examined the hepatotherapeutic effects of MSCs treatment on rats' acetaminophen-induced hepatotoxicity. According to Lin et al. (2020), MSCs have the potential to be a treatment for kidney illness caused by toxicants. Furthermore, MSC-Exos have become an attractive cell-free treatment for chronic renal disease (Cao et al., 2022). Additionally, MSC-derived Exos have been shown by Tan et al. (2014) to have hepatoprotective properties against injury caused by toxicants.
We used MSCs in this work because of their multipotent characteristics, ease of isolation from many tissues, ease of in vitro expansion, and their extensive therapeutic potential demonstrated in clinical trials. The AD-MSCs employed in this work showed the typical morphological characteristics of MSCs, such as adhesion to the growth plates and a fibroblast-like appearance. Also, AD-MSCs were characterized by flowcytometry through positive expression of CD90 and CD105 and negative expression of CD 34. According to findings by Niyaz et al. (2012), AD-MSCs isolated from rats were negative for CD45, CD106, and MHC Class II, and positive for CD29, CD90, CD54, and MHC Class I. According to Yao et al. (2015), MSCs were found to express CD14, CD34, CD45, and CD71 at very low levels, whereas they expressed high levels of CD13, CD90, CD44, and CD105.
The present investigation clarifed the regenerative effects of MSCs on the livers and kidneys of the CTX + AD-MSCs-treated group in comparison to the CTX group. The results showed improvements in the shape and arrangement of cells, as well as an inhibition of the infiltration of mononuclear inflammatory cells, at the level of histopathological examination. Additionally, improvements were observed at the level of biochemical investigations (ALT, AST, MDH, GLDH, urea, creatinine, KIM-1, and clusterin) where liver and kidney functions were restored. The protective effects of MSCs also appeared with decreased levels of MDA, TNF-α in the treated group; also the reaction and distribution of COX-2 and iNOS were decreased. Furthermore, on the molecular level, there were upregulation of Nrf-2/HO-1 and downregulation of NF-κB/TLR-4 and Bax/Bcl-2 signaling pathways. MSCs treatment provides a hepatotherapeutic impact on acetaminophen-induced hepatotoxicity in rats, as demonstrated by El Araby et al. (2022). The hepatoprotective impact of MSCs may be attributed to their anti-inflammatory, anti-apoptotic, and immunomodulatory properties.
The exact mechanism underlying MSCs' therapeutic potential remains unclear. These cells are thought to have other qualities that make them appealing for therapeutic uses in addition to their unique ability to differentiate, but also the release of a wide variety of bioactive substances that play a vital biological role in damage circumstances, such as chemokines, growth factors, and cytokines; this makes the properties of MSCs in vivo an issue of therapeutic concerns (da Silva Meirelles et al., 2009). Another possibility is that MSCs could repair damaged cells by releasing microvesicles that include proteins, mRNAs, or microRNAs (Barnes et al., 2016). However, as previously indicated, disadvantages of MSCs have limited their clinical usage. Therefore, different MSC-based and non-complicationous treatment approaches are required.
Using a conventional procedure previously outlined, the exosomes were separated and purified from AD-MSCs. Exosomes were identified by electron microscope analysis as being spheroids or cup-shaped particles. The AD-MSC Exos's particle size is less than 200 nm, according to protein content analysis. Hu et al.'s (2021) findings are similar to these data.
Comparing the liver and kidneys of the CTX + MSCs Exo-treated group to those of the CTX group and the CTX + AD-MSCs group, the results further clarified the curative properties of MSC-derived exosomes; more significant results were obtained from the regeneration effect of MSCs-Exo than from AD-MSCs alone. At the histopathological level infiltration of mononuclear inflammatory became low with decreased pyknosis in the epithelial lining of certain renal tubules and minor congestion in the hepatic sinusoids. Biochemical analyses revealed that the CTX + AD-MSCs group had significantly lower levels of ALT, AST, MDH, GLDH, urea, creatinine, KIM-1, and clusterin. The CTX + AD-MSCs-Exo group experienced a greater reduction in MDA and TNF-α levels compared to the AD-MSCs alone group. Additionally, immunohistochemical reactions demonstrated that the distribution of iNOS and COX-2 was reduced. Furthermore, on a molecular genetic level, the Nrf-2/HO-1 signaling pathway was elevated, and the NF-κB/TLR-4 and Bax/Bcl-2 signaling pathways were downregulated more strongly in the CTX + AD-MSCs-Exo group compared to the CTX + AD-MSCs group. MSC-Exos have been shown by Wang et al. (2022) to activate proliferative and regenerative responses, which may mitigate acute and chronic liver injury. Additionally, MSC-Exos have shown promise as a cell-free treatment for chronic kidney disease, as demonstrated by Cao et al. (2022).
MSC-Exos have the ability to repair tissue by stimulating angiogenesis, dedifferentiation, and cell proliferation while also reducing oxidative stress and apoptosis (Harrel et al., 2020). MSC- Exos replicate the functions of their originator MSCs through delivery of several genetic and protein cargos to the target cells (Cao et al., 2022). MiRNA cargos (like miRNA- 10a, miRNA-486) were regarded as pro-regenerative miRNAs due to their ability to promote cell proliferation (Tapparo et al., 2019) while miRNA-199a-3p was discovered to reduce apoptosis by downregulating genes linked to apoptosis (Zhang et al., 2020). Protein cargos (such as extracellular matrix metalloproteinase inducer (EMMPRIN) and metalloproteinase-9 (MMP-9)) have been found to stimulate angiogenesis (Abu El-Asrar et al., 2017). Furthermore, MSC-Exos mitigate inflammatory responses by reducing invasion of immune cells such as macrophages, T cells, and NK cells (Harrell et al., 2019). For example, cytokines including IL-6, IL-10, and hepatocyte growth factor (HGF), as well as miRNA-155 (Pers et al., 2021) and miRNA-146a (Tavasolian et al., 2021), contribute to MSC-Exos mediated immunoregulation (Wu et al., 2019). Exos therapy also offers unique benefits: it doesn't require engraftment, which lowers the risk of cancer (Tracy et al., 2019); additionally, because of its nanoscale level, it improves the penetration of barriers, biomembranes, and vasculature (Chen et al., 2016). Collectively, Exos recapitulate to a large extent the immensely broad therapeutic actions previously linked to MSCs (Phinney et al., 2017).