PTH decreases membrane expression of NaPi-2a by phosphorylation of serine-77 (S77) in the Na+/H+ exchange regulatory Selleck BTK inhibitor cofactor (NHERF)-1,

a scaffolding protein, leading to internalization and degradation of NaPi-2a [21] and [22]. A recent study suggested that the phosphaturic action of FGF23 may also involve phosphorylation of NHERF-1 at S77 [23]. To test this possibility in vivo, we injected mice with rFGF23, and performed reciprocal co-immunoprecipitation of the NaPi-2a/NHERF-1 complex on renal brush border membrane vesicle (BBMV) preparations. rFGF23 caused an almost 4-fold increase in urinary phosphate excretion compared to vehicle injection (46.9 ± 10.4 vs. 12.3 ± 0.6 mmol/mmol creatinine in vehicle-treated controls, P < 0.05), and this effect was accompanied by hypophosphatemia (2.2 ± 0.2 vs. 3.4 ± 0.3 mmol/l in vehicle-treated controls, P < 0.05). The rFGF23-induced phosphaturia was associated with a 50% reduction in membrane abundance of the NaPi-2a/NHERF-1 complex ( Fig. 4A). Immunofluorescence staining of paraffin sections with anti-NHERF-1

Navitoclax and anti-phosphoserine antibodies showed a clear reduction in the apical membrane abundance of NHERF-1, and a general increase in serine phosphorylation in proximal tubular epithelium of rFGF23-treated mice ( Fig. 4B). Moreover, the co-staining also suggested increased serine phosphorylation of NHERF-1 in the apical membrane, 8 h after rFGF23 Suplatast tosilate injection ( Fig. 4B). Collectively, these data show that FGF23 signaling targets the NaPi-2a/NHERF-1 complex in the apical cell membrane of renal tubular epithelium in vivo. Next, we examined whether FGF23 induces phosphorylation of NHERF-1 and whether SGK1 is involved in this process in isolated proximal tubular segments. Similar to PTH, a 2-hour treatment of proximal tubules with rFGF23 led to a marked increase in serine phosphorylation of NHERF-1 (Fig. 4C). Importantly, FGF23- but not PTH-induced phosphorylation of NHERF-1 could be completely blocked by an SGK1 inhibitor (Fig. 4C), suggesting

that activated SGK1 mediates phosphorylation of NHERF-1. To confirm the functional role of Klotho in FGF23 signaling, we isolated proximal tubular segments from 3-month-old wild-type, VDR∆/∆, and Kl−/−/VDR∆/∆ mice, and treated them for 2 h with 1, 10, and 100 ng/ml rFGF23 in vitro. We chose Kl−/−/VDR∆/∆ mice as αKlotho deficient animal model, because Kl−/− mice are characterized by severe alterations in mineral homeostasis [11] which might affect the results of subsequent ex vivo experiments. Kl−/−/VDR∆/∆ mice on rescue diet are normocalcemic and normophosphatemic, and have unchanged PTH serum levels compared with VDR∆/∆ mice [11]. rFGF23 treatment resulted in a similar dose dependent down-regulation of NaPi-2a expression in proximal tubular segments from wild-type and VDR∆/∆ mice ( Fig. 5), showing that this effect is vitamin D independent.