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. 2021 Dec;32(12):3051–3065. doi: 10.1681/ASN.2021020276

Figure 2.

Figure 2.

Klotho shedding responds to CaSR activation in minced kidney cortex and HEK-293 cells transiently transfected with CaSR and Klotho. (A) Klotho shedding by minced kidneys, in response to added CaSR inhibitors (10 mM EDTA or 50 μM calcilytic NPS-2143), CaSR activators (4.0 mM Ca [high Ca2+] or 10 μM calcimimetic R-568), or protease inhibitors (100 μM TAPI-1 or 2 μM GI254023X in the presence of high Ca2+). Shed Klotho responses are expressed as mean±SEM fold increases over average control values (vehicle, dashed line): EDTA, 0.57±0.09; NPS-2143, 0.91±0.04; high Ca2+, 1.61±0.11; R-568, 1.31±0.07; TAPI-1, 0.85±0.03; GI254023X, 0.88±0.08 (n=3–9). R-568 was significantly increased compared with control, NPS-2143, GI254023X, and TAPI-1. (B) Klotho shedding by HEK-293 cells expressing the CaSR and Klotho, in response to CaSR inhibitors (CaSRR795W nonfunctional mutant transfected, or 25 μM calcilytic NPS-2143), CaSR activators (4 mM Ca [high Ca2+] or 1 μM calcimimetic R-568), or protease inhibitors (100 μM TAPI-1 or 2 μM GI254023X in the presence of high Ca2+). Shed Klotho responses are expressed as mean±SEM fold increases over average control values: control, 1.0; CaSRR795W, 0.82±0.08; NPS-2143, 0.65±0.18; high Ca2+, 2.91±0.37; R-568, 1.48±0.10; TAPI-1, 0.20±0.05; GI254023X, 0.53±0.04. R-568 was significantly increased compared with control, CaSRR795W, NPS-2143, GI254023X, and TAPI-1. **P<0.01, ***P<0.001, ****P<0.0001 by ANOVA and multiple comparisons post-test. (C) Klotho shedding by minced kidney in response to HEPES-buffered solution supplemented with 250 μm R-568 or 250 μm NPS-2143 at indicated pH values. Shed Klotho responses are expressed as mean±SEM fold increases over vehicle (Veh) at pH 7.0: for pH 7.0: Veh, 1.0; R-568, 1.16±0.05; NPS-2143, 1.15±0.02; for pH 7.4: Veh, 1.34±0.04; R-568, 1.40±0.07; NPS-2143, 1.04±0.12; for pH 7.8: Veh, 1.65±0.14; R-568, 2.01±0.32; NPS-2143, 1.20±011. (D) Klotho shedding by HEK-293 cells expressing the CaSR and Klotho in response to pH of DMEM (HCO3 buffer) pre-equilibrated in humidified incubators with CO2 content of 20% (pH 7.0) and 5% (pH 7.7). Values are expressed as mean±SEM fold change from standard DMEM which contains 1.6 mM Ca, pH 7.7. Values expressed as mean±SEM are: for pH 7.0: Ca 1.6 mM, 0.57±0.09; Ca 4 mM, 1.22±0.15; Ca 4 mM and NPS-2143, 0.54; for pH 7.7: Ca 1.6 mM, 1; Ca 4 mM, 2.7±0.34; Ca 4 mM and NPS-2143, 0.72±0.15; Ca 4 mM and GI245023X, 0.53±0.04. *P<0.5 compared with pH 7 vehicle treated, ***P<0.001 compared with pH 7.7 vehicle treated, ++P<0.01, ++++P<0.0001 compared with 4 mM high Ca at pH 7.7, as determined ANOVA, with multiple comparisons post-test.