Abstract
Background and Aims
Cisplatin, is a nonspecific cytotoxic agent that primarily interferes with cellular DNA replication and the cell cycle, nevertheless it lacks tumor selectivity and acts also in normal cells. The most serious adverse reaction of cisplatin is Acute Kidney Injury (AKI), limiting its use and efficacy in chemotherapy. Cisplatin nephrotoxicity is observed in more than 30% of older patients, however the mechanism of nephrotoxicity remains unclear and specific preventive measures are not available. Today, there is an urgent need for specific nephroprotective strategies to be used during cisplatin chemotherapy. Recently, we found that tubular stem/progenitor cells (tARPC) are able to protect the tubular epithelial (RPTEC) from cisplatin induced injury, preserving their proliferation and inhibiting apoptosis. The aim of this study was to identify the molecular mechanisms involved in tARPC-mediated resistance to cisplatin.
Method
Co-cultures of RPTEC cells and tARPCs were exposed to cisplatin (2.5 µM) for 6 h and then kept in culture for 96 h. Gene expression profile was obtained from tARPCs and RPTECs by Agilent SurePrint G3 Human Gene Expression Microarrays. Genespring and R software were used for the analysis. Gene expression data were validated by Real-time PCR. Extracellular vesicles were isolated from cell culture supernatant by miRCURY Exosome Cell/Urine/CSF Kit (Qiagen) and RNA contained in extracellular vesicles was purified, analyzed in quality by Bioanalyzer (RNA nano) and evaluated by qPCR. The BrdU assay and caspase 3 were used to measure proliferation and apoptosis levels. Immunohistochemical expression of activated caspase-3 was used as a marker of apoptosis in RPTECs.
Results
By a whole-genome gene expression analysis, we found 107 genes specifically modulated by RPTECs in response to cisplatin and, among these, 30 genes induced by ARPCs following the cisplatin damage. In particular, we found a strong upregulation of the CYP1B1 gene (false discovery rate corrected p value <0.05; fold change=4,1). The qPCR confirmed the increase in CYP1B1 levels in the co-cultures with respect to the respective basal conditions (p <0.05). Interestingly, the CYP1B1 mRNA was also enveloped in Extracellular Vesicles released in the cell co-culture media by tARPC and RPTEC after cisplatin exposition. The CYP1B1 gene encodes a member of the cytochrome P450 superfamily of enzymes and the produced enzyme metabolizes procarcinogens, such as polycyclic aromatic hydrocarbons. CYP1B1 has been shown to be active within tumors and is also capable of metabolizing a structurally diverse range of anticancer drugs. It is responsible for the resistance to docetaxel, cisplatin, tamoxifen and nucleoside analogues. CYP1B1 is involved in the detoxification of the body by various exogenous toxic agents, including cisplatin. We found that CYP1B1 gene was expressed at low levels in RPTECs and in cisplatin-damaged RPTECs. Moreover, 96 h days after 2.5 μM exposure to cisplatin, RPTECs reduced the proliferation and underwent in apoptosis, as showed by caspase 3. However, in co-culture with ARPCs, ARPC cellular and extracellular vesicles CYP1B1 gene expression significantly increased, the apoptotic process was stopped and RPTECs increased their proliferation rate.
These data support the hypothesis that ARPCs are sensor of cisplatin damaged-RPTEC and confers cisplatin resistance by transferring CYP1B1 gene in extracellular vesicles.
Conclusion
This is the first evidence of a cisplatin-induced overexpression of CYP1b1 in renal epithelial cells as a defense mechanism against cisplatin toxicity. This is consistent with our previous data showing that renal progenitors are resistant to cisplatin. The findings may have biological and clinical significance in terms of their implications in cellular communications and potential use of CYP1B1 as biomarkers for AKI induced by cisplatin or as protective agent.
Drug Dosing Rationality Based on Jelliffe Equation in Acute Kidney Injury
