Effection Of QDPR Gene Mutation And Expression On BH₄–Ralated Enzymes Of NRK-52E Cells Under High Glucose

Objectives By studying whether the changes of quinoid dihydropteridine reductase(QDPR) protein expression level and K93 T mutation can affect the BH4-relating enzymes(DHFR, TPH1, TH and PAH) in NRK-52 E cell under high glucose ambience, explore the probable underlying mechanism of QDPR gene in diabetic nephropathy(DN).Methods The expressions of DHFR, PAH, TPH1 and TH proteins in rat kidney of type 2 Diabetic Nephropathy were examined by immunocytochemical stain. Construct the lentivirus of QDPR protein overexpression, K93 T mutation and QDPR gene knockdown. The lentivirus were transfected into NRK-52 E cells, and the cell models of QDPR overexpression, K93 T mutation and knockdown were established. Then western blot and real-time PCR were performed to identify the expression levels of QDPR protein and m RNA. The cell models of QDPR protein overexpression, K93 T mutation and knockdown were cultured with the normal glucose(5.4mmol/L) and high glucose(30mmol/L) ambience separately for 72 h. The experients are grouped as follows, 1 NRK-52 E Control,(NC) 2 NRK-52 E exposed to High glucose,(NHG) 3 overexpression lentivirus control,(LV-OCON) 4 overexpression lentivirus control exposed to High glucose,(LV-OCON-HG) 5 lentivirus overexpressed QDPR,(LV-QDPR) 6 lentivirus overexpressed QDPR exposed to High glucose,(LV-QDPR-HG) 7 lentivirus overexpressed K93 T QDPR,(K93T-QDPR) 8 lentivirus overexpressed K93 T QDPR exposed to High glucose,(K93T-QDPR-HG) 9 lentivirus SHDNA control,(LV-SHCON) 10 lentivirus SHDNA exposed to High glucose,(LV-SHCON-HG) 11 lentivirus QDPR SHDNA,(LV-SHQDPR) 12 lentivirus QDPR SHDNA exposed to High glucose,(LVSHQDPR-HG). Western blot was performed to identify the expression of the BH4-relating enzymes(DHFR, TPH1, TH and PAH) in NRK-52 E cell under high glucose ambience, exploration the probable underlying mechanism of QDPR gene in diabetic nephropathy(DN).Results 1 DHFR, TPH1 and PAH protein express in rat kidney tubule, but TH protein does not express. 2 The western blot revealed that DHFR, TPH1 and PAH protein decreased in NHG group when compared with NC group in NRK-52 E cells. 3 In high glucose ambience, compared with LV-OCON-HG group, the protein expressions of DHFR was significantly decreased(P < 0.001), TPH1 was decreased(P < 0.05) and PAH was decreased(P < 0.01) in LV-QDPR-HG group, but TH protein does not express in NRK-52 E cells with QDPR overexpression. 4 The comparison of DHFR, TPH1 and PAH expression levels between LV-SHQDPR-HG and LV-SHCON-HG group showed no difference, but TH protein does not express in NRK-52 E cells with QDPR knockdown. 5 In high glucose ambience, compared with LV-QDPR-HG group, the protein expressions of DHFR was increased(P < 0.05), TPH1 was significantly increased(P < 0.01) and PAH was increased(P < 0.05) in K93T-QDPR-HG group, but TH protein does not express in NRK-52 E cells with QDPR K93 T mutation.Conclusions 1 The expressions of DHFR, TPH1 and PAH protein decreased in NRK-52 E cells of high glucose ambience, these results suggest that DHFR, TPH1 and PAH protein play an important role in the development of DN. 2 The changes of QDPR protein expressions and K93 T mutation lead to changes the content of BH4-relating enzymes(DHFR, TPH1, TH and PAH), which implies that QDPR gene influences the occurrence and process of DN by regulating BH4-relating enzymes.