Measurement of Lipogenic Flux in Kidney Using Deuterium Oxide (D2O)

Background: Diabetes is the leading cause of end stage kidney disease in the United States. Previous studies in our laboratory using stable isotope have shown that hyperglycemia leads to increased de novo lipogenesis (DNL) in kidney cells.Though lipogenic 13C-substrates detect DNL with high sensitivity, quantification is complicated. Here we show that the stable isotope Deuterium (2H) can be used both in vitro and in vivo to overcome these challenges. D2O treatment enables labeling of most biomolecules making it ideal for multiomic study. Administration of D2O to biological systems is simpleand safe for longer time periods. Methods: We developed a method using LC/ /MS to measure 2H labeling in target metabolites. Deuterium incorporation is measured as mass shifts in the spectra (m+2, m+3). The lipogenic flux is measured as the percentage of 2H labeling in palmitate, the primary product of FASn. Results: Time and dosimetry studies in HK-2 cells show that incubating cells in media with 25% D2O for 48 hours is optimum to study lipogenic flux. DNLactivity in human podocyte under normal (NG) and hyperglycemic (HG) conditions was examined by incubating cells in D2O media with 5.5 mM or 25 mM glucose for 48 hours. As we have shown in our previous studies using 13C isotope, cells grown under hyperglycemic conditions show an enhanced 2H labeling of palmitate suggesting increased lipogenic flux (Fig A). For in vivo lipogenic flux studies mice with pathological features of DN (BKS db/db) and control (db/+) were given D2O (20ul/g BW) IP at 0 hour and 24 hour then sacrificed at 48 hour. Total lipids were extracted from kidney cortex and 2H labeling was analyzed. Our results show higher 2H labeling in diabetic mice kidney cortex (m+3, m+4 and m+5) (Fig B) indicating increased lipogenic flux. Conclusion: These results show that 2H labeling with LC/MS can be used to study lipogenic flux in target tissues both in vivo and in vitro. Funding: Private Foundation SupportResults of in vitro and in vivo Deuterium labeling in kidney cells.