Fig. 5: MSCs reversed cytokine-mediated mitochondrial dysfunction in TECs.

Conditioned media from RAW264.7 cells (see Fig. 3.) was transferred to HK-2 cells for 24 h. a Effect of MSCs on the mRNA (left panel) and protein expression (right panel) of the mitochondrial biogenesis markers PGC-1α, mtTFA, and COX-IV in HK-2 cells. b Effect of MSCs on mitochondrial respiration. Real-time OCRs were measured using an XF24 extracellular flux analyzer. During the measurements, 1 μg/ml oligomycin (Oligo), 1 μm carbonyl cyanide p-(trifluoromethoxy)-phenyl-hydrazone (FCCP), and 1 μm rotenone (Rote) plus 2 μm antimycin A (AA) were sequentially added. c The area under the curve of the basal OCR (left panel) and the ECAR (right panel). All OCRs and ECARs were normalized based on the cell number. Data show the means ± SEMs (n = 5). ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 versus control (cRAW → HK-2); ^#P < 0.05 and ^##P < 0.01 versus LPS-treated cells (aRAW → HK-2). d MSC treatment increased the mitochondrial mass. Mitochondria stained with 100 nm MitoTracker Red are shown in red, and nuclei stained with DAPI are shown in blue. The treatment of HK-2 cells with conditioned media from aRAW cells significantly decreased mitochondrial mass. In contrast, the coculture of MSCs with conditioned media from aRAW cells increased mitochondrial mass. Scale bars, 10 µm (n = 4). e MSCs decreased mitochondrial ROS production in HK-2 cells, as measured using MitoSOX-based flow cytometry (n = 4). cRAW, untreated RAW264.7 cells; aRAW, LPS-treated RAW264.7 cells; aRAW + MSCs, aRAW cells cocultured with MSCs. aRAW → HK-2, HK-2 cells were treated with conditioned media from aRAW cells; aRAW + MSCs → HK-2, HK-2 cells treated with conditioned media from aRAW cells cocultured with MSCs