Evaluating RNA Oxidation In Diabetic Nephropathy And Exploring Its Mechanisms

An enormous public health burden is caused by Diabetes mellitus(DM)since diabetic complications often lead to disability and death.Diabetic nephropathy(DN)is the most common microvascular complications of diabetes and the strongest predictor of mortality in patients with diabetes.Oxidative stress caused by increased free radical production is believed to play a central role in the development of diabetes and diabetic complications.RNA undergoes significant oxidative damage,and RNA oxidation is viewed as early stage at which the clinical symptoms are very discrete in some diseases.There are now convincing data suggesting that RNA oxidation is a mechanism for disease development.It is recently found that urinary 8-oxo-Gsn,marker of RNA oxidation,is also an independent predictor of mortality in patients with type 2 diabetes,however,there is little additional information.Hyperproinsulinemia is a common characteristic of type 2 diabetes.It is also associated with the development of diabetic complications.Although increased ROS caused by environmental factors may induce hyperproinsulinemia,which would finally lead to type 2 diabetes,the detailed mechanism remain elicited.A growing list of insulin gene mutations may cause a new form of monogenic diabetes and hyperproinsulinemia,but there is few insulin gene mutation in type 2 diabetes.Based on there are widespread RNA and DNA sequence differences in the human transcriptome and our previous results that RNA oxidation may produce abnormal proteins,it is important to find out whether RNA oxidation can induce abnormal proinsulin and lead to diabetes eventually.Methods and resultsFirstly,we used a sensitive and accurate method based on isotope dilution high-performance liquid chromatography-triple quadruple mass spectrometry(ID-LC-MS/MS)to determine the levels of 8-oxo-7,8-dihydro-2-deoxyguanosine(8-oxo-dGsn)and 8-oxo-7,8-dihydroguanosin(8-oxo-Gsn)in various tissue specimens,plasma and urine of hyperglycemic SD rats induced by streptozotocin(STZ).The oxidative DNA and RNA damage were observed in various organs and the amounts of 8-oxo-dGsn and 8-oxo-Gsn derived from DNA and RNA were increased with hyperglycemic status.The levels of 8-oxo-Gsn in urine and plasma were significantly higher compared to that of 8-oxo-dGsn,which most likely reflected the RNA damage that occurs more frequently compared to DNA damage.For the oxidative stress induced by hyperglycemia,8-oxo-Gsn in urine may be a sensitive biomarker on the basis of the results in urine,plasma and tissues.Secondly,to evaluate DNA and RNA oxidation in early stage of diabetic nephropathy,we analyzed the oxidatively generated guanine nucleosides in urine and renal tissue from different age db/db mice.It was found that the levels of 8-oxodGsn and 8-oxo-Gsn were increased in urine and renal tissue of db/db mice,and db/db mice with early symptoms of diabetic nephropathy suffered from more extensive oxidative damage compared with lean littermate control db/m mice.Importantly,increased levels of 8-oxo-dGsn or 8-oxo-Gsn are paralleled changes of diabetic nephropathy in db/db mice.In addition,the amounts of the free forms of 8-oxo-dGsn or 8-oxo-Gsn in urine were in good agreement with levels of renal 8-oxo-dGsn/106dGsn or 8-oxo-Gsn/106Gsn respectively,and 8-oxo-Gsn is higher in renal tissue and urine compared with 8-oxo-dGsn.Thirdly,we established an absolute quantitative detection method based on liquid chromatography electrospray ionization mass spectrometry to determine anbnormal proinsulin.Finally,we established different cell lines which could produce proinsulin or abnormal proinsulin using lentiviral expression system.ConclusionHyperglycemia-induced oxidative stress could induce DNA/RNA oxidation in different organs.Although the susceptibility to oxidative damage is various from organs,the levels of DNA/RNA oxidation increase with the duration of hyperglycemia,and RNA oxidation is more significant.Hyperglycemia-induced DNA/RNA oxidation in kidney is more significant compared with other organ.There is a relationship between diabetic nephropathy and DNA/RNA oxidation.Compared with DNA oxidation,RNA oxidation is more closely associated with early change of diabetic nephropathy.Urinary 8-oxo-Gsn could be a novel,noninvasive,and easily detected biomarker for diabetes and diabetic kidney diseases.Establishments of absolute quantitative detection method and cell lines would be useful for further studies to find out whether RNA oxidation induced abnormal proinsulin may eventually lead to diabetes.