Isorhamnetin Attenuates Hyperglycemia-Induced Diabetic Nephropathy-Like Alterations in Human Embryonic Kidney Cells (HEK-293): In vitro Insights into the Potential Role of FOXO1

Background

Diabetic nephropathy is becoming an alarming concern globally as the percentage of patients is increasing day by day. Although the suffering rate was too high, the existing treatment was very limited and non-targeted.

Objectives

To overcome this condition, the plant flavonoid Isorhamnetin (ISN) was used in this study to investigate its protective effect on hyperglycemia-mediated diabetic nephropathy.

Materials and Methods

Cytotoxicity and nephroprotective potency of Isorhamnetin were investigated with the help of a Tetrazolium-based assay. Apoptosis was confirmed by Acridine orange/Ethidium Bromide (AO/EtBr) dual staining and comet assay, where DNA damage was investigated in both methods. Mitochondrial membrane potential was also assessed by the Mito potential kit. Oxidative stress status was analysed by checking the release of Reactive Oxygen Species (ROS) and reduced Glutathione (GSH). The molecular mechanism also involved the expression of FOXO1, which was investigated between the hyperglycemic condition and the isorhamnetin-treated condition.

Results

Isorhamnetin exhibited an IC₅₀ value of 93.50 µg/mL, but at low dosage (6.25 µg/mL), it exhibited a great impact on the high glucose-induced HEK 293 cells. A similar trend was continued in dual staining as well as in mitochondrial membrane potential assessment, as isorhamnetin ameliorates the deterioration effect created by the hyperglycemic condition. Isorhamnetin elevated the antioxidant enzyme release (GSH) and pulled down the free radicals generated by high glucose exposure. The FOXO1 gene was downregulated in the hyperglycemic group, which was reversed in the isorhamnetin-treated group.

Conclusion

Thus, the evidence proved that Isorhamnetin provides beneficial positive impacts on the diabetic nephropathy condition. Since there are very few studies, the in vivo investigation further adds depth to molecular knowledge about detailed signaling pathways.