Fig 2: The expression of UMOD in oxalate-induced NRK-52E cells and its physiological interaction with TRPV5. (a) QRT-PCR was performed to detect the mRNA expression of UMOD in oxalate-induced NRK-52E cells. ß-Actin served as the internal control. (b) Western blot was performed to measure the protein level of UMOD in oxalate-induced NRK-52E cells. ß-Actin served as the internal control. (c) CoIP was used to detect the physiological interaction between TRPV5 and UMOD. (d) After UMOD overexpression in NRK-52E cells, qRT-PCR was used to verify the expression of UMOD. ß-Actin served as the internal control. (e) The UMOD protein level was verified using Western blot. ß-Actin served as the internal control. **p < 0.01 and ***p < 0.001 vs. control; ###p < 0.001 vs. NC. UMOD: uromodulin; qRT-PCR: quantitative real-time polymerase chain reaction; CoIP: coimmunoprecipitation; NC: negative control.

Fig 3: miR-103a-3p silencing activated the UMOD/TRPV5 axis to attenuate NRK-52E cells from oxalate-induced injury. (a) The effect of the miR-103a-3p inhibitor on cell viability in NRK-52E cells treated with oxalate, TRPV5-overexpressing plasmid, and siUMOD was determined by MTT assay. (b) Crystal cell adhesion assay was used to quantify CaOx deposition in specified cells (×200 magnification, scale bar = 200 µm). (c) The Ca2+ content measurement in different groups was analyzed by the Fluo-4 AM Kit. ***p < 0.001 vs. oxalate; ###p < 0.001 vs. oxalate + TRPV5 + siNC + IC; ??p < 0.01 and ???p < 0.001 vs. oxalate + TRPV5 + siUMOD + IC. TRPV5: transient receptor potential cation channel subfamily V member 5; UMOD: uromodulin; MTT: methylthiazolyldiphenyl-tetrazolium bromide; NC: negative control; siUMOD: small interfering RNA against UMOD; I: inhibitor; IC: inhibitor control.

Fig 4: The effect of TRPV5 on oxalate-induced NRK-52E cells. (a) QRT-PCR was performed to detect the mRNA expression of TRPV5 in NRK-52E cells after TRPV5 overexpression. ß-Actin served as the internal control. (b) Western blot was performed to measure the protein level of TRPV5 in NRK-52E cells after TRPV5 overexpression. ß-Actin served as the internal control. (c) The expression of TRPV5 in oxalate-induced NRK-52E cells with or without TRPV5 overexpression was detected by qRT-PCR. ß-Actin served as the internal control. (d) The protein level of TRPV5 in oxalate-induced NRK-52E cells with or without TRPV5 overexpression was measured by Western blot. ß-Actin served as the internal control. (e) MTT assay was conducted to evaluate cell viability. (f) Crystal cell adhesion assay was used to determine and quantify CaOx deposition in specified cells (×200 magnification, scale bar = 200 µm). ***p < 0.001 vs. NC; ???p < 0.001 vs. control; #p < 0.05 and ###p < 0.001 vs. oxalate + NC. TRPV5: transient receptor potential cation channel subfamily V member 5; qRT-PCR: quantitative real-time polymerase chain reaction; MTT: methylthiazolyldiphenyl-tetrazolium bromide; NC: negative control.

Fig 5: 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. ß-Actin served as the internal control. (c) Western blot was performed to verify the protein level of UMOD after siUMOD transfection. ß-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 bar = 200 µm). (f) The Ca2+ content measurement in different groups was analyzed by Fluo-4 AM Kit. ***p < 0.001 vs. siNC; &&&p < 0.001 vs. control; ##p < 0.01 and ###p < 0.001 vs. oxalate; ??p < 0.01 and ???p < 0.001 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.