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. 2018 Apr 11;13(4):e0189464. doi: 10.1371/journal.pone.0189464

Fig 11. Bicarbonate-dependent pH recovery assays in cultured hRPTCs carrying Wild-Type (WT) or homozygous variant (HV) SLC4A5.

Fig 11

A) Bicarbonate-dependent pH recovery: pH recovery was measured in WT and HV SLC4A5 hRPTCs expressing empty vector (vector control, VC), overexpressing (OE) SLC4A5 (4A5OE), knock-down (KD) SLC4A5 (4A5KD), and knock-down (KD) SLC4A4 (4A5KD). Bicarbonate-dependent pH recovery is faster in SLC4A5 OE (4A5OE) (*P<0.001 and &P<0.01) and slower in SLC4A5 KD (4A5KD) (**P<0.001 and &&P<0.001), relative to VC in both WT and HV SLC4A5 hRPTCs. By contrast, bicarbonate-dependent pH recovery is not altered in SLC4A4 KD, relative to VC in either WT or HV SLC4A hRPTCs. Monensin (10 μmol/L, 24 h) treatment that increases intracellular sodium (↑Na+) increases pH recovery rate in HV but not WT SLC4A5 hRPTCs (#P<0.001). In SLC4A5 OE (4A5OE) hRPTCs, pH recovery rate is increased by monensin (10 μmol/L/24 h) (↑Na+) in both WT (##P<0.001) and HV SLC4A5 ($ $P<0.001) but to a greater extent in the latter than in the former ($P<0.001). Knockdown of SLC4A5 (4A5KD) prevents the stimulatory effect of monensin on pH recovery rate in both WT and HV SLC4A5. Knockdown of SLC4A4 (4A4KD) does not affect the increased pH recovery in monensin-treated (↑Na+) HV SLC4A5 hRPTCs (%P<0.05 HV 4A4KD vs WT 4A4KD). B) Increased bicarbonate-dependent pH recovery rate in monensin-treated HV SLC4A5 hRPTCs is blocked by HNF4A inhibitors. Bicarbonate-dependent pH recovery was measured in vehicle (VEH) or monensin (10 μmol/L, 24 h)-treated (↑Na+) HV SLC4A5 hRPTC in the presence of HNF4A inhibitors BIM5078 or BI6015. These inhibitors have no effect when added alone, but either inhibitor completely blocks (n = 4 #P<0.05 vs monensin HV, two-way ANOVA, Holm-Sidak test) the monensin-stimulated (↑Na+) (n = 12 *P<0.05, two way ANOVA, Holm-Sidak test) increase in bicarbonate-dependent pH recovery rate in HV hRPTCs.