FOXO1 Overexpression Attenuates Tubulointerstitial Fibrosis and Apoptosis in Diabetic Kidneys by Ameliorating Oxidative Injury via TXNIP-TRX

Figure 6

TXNIP-TRX expression in HK-2 cells cultured under high glucose. (a) Expression of FOXO1 and p-FOXO1 detected by RT-PCR. FOXO1 knockin (KI) and knockout (KO) HK-2 cells were established with CRISPR/Cas9 and cultured under high glucose (HG; 4.5 g/L glucose) or normal glucose (NG; 1.0 g/L glucose) conditions. (b) Representative immunoblots. (c, d) FOXO1 and ratio of p-FOXO1/total FOXO1 as determined by densitometric analysis. (e–h) mRNA and protein levels of TXNIP and TRX detected by quantitative RT-PCR analysis of total RNA and western blot, respectively. FOXO1 KI cells were treated with the TRX inhibitor PX-12, and FOXO1 KO cells were treated with a small interfering RNA against TXNIP (si-TX). (i–k) The fibrosis- and apoptosis-related proteins, (i) FN, (j) COL IV, and (k) BAX, were detected by western blot analysis. (l–o) Chromatin immunoprecipitation assays showing FOXO1 binding to the promoter regions of TXNIP and TXN in HK-2 cells under HG. Soluble chromatin was immunoprecipitated with antibodies against FOXO1. The DNA fragments were analyzed by qPCR (l, m) or amplified by PCR and visualized on agarose gels (n, o). (p) Overexpression of FOXO1 prevents reactive oxygen species (ROS) accumulation in HG-treated HK-2 cells. Intracellular ROS production was quantified by flow cytometry analysis using 2,6-dichlorofluorescein diacetate. The data are presented as the (). vs. normal glucose (NG); vs. HG; vs. FOXO1 knockin (KI); vs. FOXO1 knockout (KO).

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