miR-103a-3p Silencing Ameliorates Calcium Oxalate Deposition in Rat Kidney by Activating the UMOD/TRPV5 Axis

Figure 3

The regulating role of the UMOD/TRPV5 axis in oxalate-induced NRK-52E cells. (a) Cell-surface biotinylation assay was applied to assess the enrichment of TRPV5 on the surface of NRK-52E cells after UMOD overexpression. (b) QRT-PCR was used to verify the mRNA expression of UMOD after siUMOD transfection. β-Actin served as the internal control. (c) Western blot was performed to verify the protein level of UMOD after siUMOD transfection. β-Actin served as the internal control. (d) MTT was used to measure cell viability following the treatment with oxalate, TRPV5-overexpressing plasmid, and siUMOD in NRK-52E cells. (e) Crystal cell adhesion assay was used to determine and quantify CaOx deposition in specified cells (×200 magnification, scale ). (f) The Ca²⁺ content measurement in different groups was analyzed by Fluo-4 AM Kit. vs. siNC; vs. control; and vs. oxalate; and vs. oxalate + TRPV5 + siNC. TRPV5: transient receptor potential cation channel subfamily V member 5; UMOD: uromodulin; qRT-PCR: quantitative real-time polymerase chain reaction; MTT: methylthiazolyldiphenyl-tetrazolium bromide; NC: negative control; siUMOD: small interfering RNA against UMOD.

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