Cyclophosphamide: Potential Hepatorenal Toxicity and the Possible Therapeutic Role of Mesenchymal Stem Cell-Derived Exosomes in Wistar Rats

Abstract

Background: Cyclophosphamide (CTX) is an alkylating agent widely described in management of several non-neoplastic and neoplastic disorders. The most observed adverse consequence of CTX is organ damage. Exosomes derived from mesenchymal stem cells (MSCs-Exos) have been shown to exhibit therapeutic effects in various tissue-injury models. Aim: The aim of this work was to examine impact of AD-MSCs-Exos in a rat model of hepatorenal toxicity. Methods: 32 rats were grouped into 4 groups (n=8): Control group: rats received intraperitoneally (i.p.) PBS (phosphate buffered saline), CTX group: rats injected i.p. with a single dose of CTX (50 mg/kg) followed by rotating doses of 8 mg/kg of CTX daily for 2 weeks, CTX+AD-MSCs group: rats infused with (1×106 AD-MSCs cells/rat) dissolved in PBS intravenously (i.v.) day after day for one week starting from second day of CTX last dose, and CTX+AD-MSCs-Exos group: rats injected with 100 μg of Exos derived from AD-MSCs in 1 ml PBS by i.v. injection for one week starting from second day of CTX last dose. 5 weeks following initial CTX dose, blood, liver, and kidneys were extracted. Serum ALT, AST, creatinine and urea levels; hepatic malate dehydrogenase (MDH) and glutamate dehydrogenase (GLDH); renal kidney injury molecule-1 (KIM-1) and clusterin were measured. The inflammatory molecule (TNF-α) and malonialdehyde (MDA); lipid peroxidation one were estimated in hepatic and renal tissues. Furthermore, NF-κB/TLR-4, Nrf-2/HO-1 and Bax/Bcl-2 signaling pathways were analyzed by qRT-PCR. Immunohistochemical staining for cyclooxygenase-2 "COX-2" and inducible nitric oxide synthase "iNOS" were also performed in hepatic and renal tissues. Finally, histopathological investigation of both liver and kidney tissue was carried out. Results: treatment with AD-MSCs-Exos improved liver and kidney functions, diminished oxidative stress (MDA) and enhanced antioxidative Nrf-2/HO-1 pathway; inhibited inflammatory response (TNF-α) and NF-κB/TLR-4 pathway; and downregulated apoptotic Bax/Bcl-2 signaling pathway compared to CTX and CTX+AD-MSCs treated groups. Also, immunological and histopathological investigation verified curative effect of AD-MSCs-Exos against CTX-induced hepatorenal toxicity. Conclusion: these findings uncovered therapeutic impact of AD-MSCs-Exos against hepatorenal insult from holistic perspective. The mechanisms behind this action included restoration of oxidant/antioxidant equilibrium, inhibition of inflammatory reaction and suppression of apoptotic machinery.