Liver X receptor-activating ligands modulate renal and intestinal sodium-phosphate transporters

Abstract

Cholesterol is pumped out of the cells in different tissues, including the vasculature, intestine, liver, and kidney, by the ATP-binding cassette transporters. Ligands that activate the liver X receptor (LXR) modulate this efflux. Here we determined the effects of LXR agonists on the regulation of phosphate transporters. Phosphate homeostasis is regulated by the coordinated action of the intestinal and renal sodium-phosphate (NaPi) transporters, and the loss of this regulation causes hyperphosphatemia. Mice treated with DMHCA or TO901317, two LXR agonists that prevent atherosclerosis in ApoE or LDLR knockout mice, significantly decreased the activity of intestinal and kidney proximal tubular brush border membrane sodium gradient-dependent phosphate uptake, decreased serum phosphate, and increased urine phosphate excretion. The effects of DMHCA were due to a significant decrease in the abundance of the intestinal and renal NaPi transport proteins. The same effect was also found in opossum kidney cells in culture after treatment with either agonist. There was increased nuclear expression of the endogenous LXR receptor, a reduction in NaPi4 protein abundance (the main type II NaPi transporter in the opossum cells), and a reduction in NaPi co-transport activity. Thus, LXR agonists modulate intestinal and renal NaPi transporters and, in turn, serum phosphate levels.

Figure 1. Effect of the LXR agonist DMHCA and TO901317 on the abundance of LXR target genes in mouse kidney and ileum

(a, b) LXR target gene mRNA abundance in kidney and ileum was analyzed by real-time quantitative PCR. TO901317 induced significant increases in the mRNA abundance of ABCA1 and ABCG1 as well as SREBP1c, FAS, and SCD1. Increases in the mRNA abundance of these genes with DMHCA were lower than activation by TO901317, especially activation of lipogenic genes, such as SREBP1c, FAS, and SCD1. DMHCA or TO901317 did not activate ChREBP and LPK, neither in kidney nor in ileum. (c) ABCA1 and SCD1 protein expression in mouse kidney was analyzed by western blotting. TO901317 induced a more significant upregulation of both of these proteins in kidney, which was also confirmed by immunofluorescence. (d, e) The expressions of ABCA1 and SCD1 were also increased in mouse ileum. Protein increase of SCD1 was not observed with DMHCA. Values represent means ± s.e.m., at least n = 6 mice per group. ChREBP, carbohydrate-responsive-element-binding protein; DMHCA, N,N-dimethyl-3β-hydroxy-cholenamide; LPK, liver pyruvate kinase; LXR, liver X receptor; NS, nonsignificant; SCD1, stearoyl-CoA desaturase 1; SREBP1c, sterol-regulatory-element-binding protein 1c.

Figure 2. Treatment with DMHCA or TO901317 reduces Na⁺-dependent phosphate uptake in mouse ileum and kidney BBM

(a) Sodium-dependent ³²P uptake was reduced in ileum BBM in DMHCA- or TO901317-treated mice. (b) Sodium-dependent ³²P was also reduced in kidney BBM in DMHCA- or TO901317-treated mice. Small panels in the upper right show sodium-independent uptake, measured in presence of Cl-choline, compared with the total phosphate uptake (NaCl). These values represent the average of two different experiments with at least n = 10 per group. BBM, brush border membrane; DMHCA, N,N-dimethyl-3β-hydroxy-cholenamide; NaPi, sodium–phosphate; Pi, inorganic phosphate.

Figure 3. Treatment with DMHCA or TO901317 decreases blood phosphate concentration, increases urine phosphate excretion in mouse, and does not change serum calcium concentration

(a) Treatment with TO901317 caused a 20% decrease in serum phosphate concentration, with a smaller reduction of 14% after treatment with DMHCA. (b) Approximately a 30% increase in urine phosphate excretion with either compound. (c) No changes in the serum calcium concentration were observed with either compound. At least n = 10 mice per group. DMHCA, N,N-dimethyl-3β-hydroxy-cholenamide; NS, nonsignificant; Pi, inorganic phosphate.

Figure 4. Effects of DMHCA and TO901317 on mouse serum FGF23 and serum PTH

(a) Treatment with TO901317 caused a 64% increase in serum FGF23 concentration, with a smaller increase of 46% after treatment with DMHCA. (b) Changes on the serum PTH levels were determined to be not significant for both compounds. At least n = 10 mice per group. DMHCA, N,N-dimethyl-3β-hydroxy-cholenamide; FGF23, fibroblast growth factor 23; NS, nonsignificant; PTH, parathyroid hormone.

Figure 5. Effects of DMHCA or TO901317 on renal BBM NaPi transporter protein abundance and NaPi transporter mRNA abundance

(a) Mouse kidney brush border membrane (BBM) vesicles were isolated after treatment with DMHCA or TO901317. A significant decrease in protein abundance was observed in all three NaPi transporters: 57% for NaPi-2a, 40% for NaPi-2c, and 30% for PiT-2. (b) NaPi transporter mRNA abundance was measured by quantitative PCR and normalized against cyclophilin A. Decreases in NaPi-2a and NaPi-2c mRNA levels were observed with either DMHCA or TO901317. (c) NHERF1 and PDZK1 protein levels were not affected by these LXR agonists. At least n = 6–8 mice per group. BBM, brush border membrane; DMHCA, N,N-dimethyl-3β-hydroxy-cholenamide; NHERF1, Na/H exchange regulatory factor-1; NaPi, sodium-phosphate; NS, nonsignificant.

Figure 6. Effects of DMHCA or TO901317 on intestinal BBM NaPi transporter protein abundance and NaPi transporter mRNA abundance

(a) Mouse ileum brush border membrane (BBM) vesicles were isolated after treatment with DMHCA or TO901317. There was a significant 61% decrease in the protein abundance of the major intestinal NaPi transporter NaPi-2b with TO901317 treatment, and a 52% after treatment with DMHCA, while there were no significant changes in type III NaPi transporter PiT1 protein expression. (b) NaPi2b mRNA abundance was reduced by 50% after treatment with DMHCA, and by 75% with TO901317. (c) NHERF1 and PDZK1 protein levels were not significantly affected by DMHCA or TO901317. At least n = 6 mice per group. BBM, brush border membrane; DMHCA, N,N-dimethyl-3β-hydroxy-cholenamide; NHERF1, Na/H exchange regulatory factor-1; NaPi, sodium-phosphate; NS, nonsignificant.

Figure 7. Treatment of opossum kidney (OK) cells with LXR agonists DMHCA or TO901317 induces activation of endogenous LXR, decrease in the phosphate uptake in a dose-dependent manner, and decrease in the expression of the endogenous NaPi transporter (NaPi4)

(a) Activation and translocation of the endogenous LXR nuclear receptor is shown by immunofluorescence after incubation of the OK cells with DMHCA. Notice the increased red signal inside the nucleus in the treated cells. (b) Correlation between concentration of the LXR agonist and reduction of the whole cells ³²P uptake was observed for both compounds. (c) Reduced expression of the endogenous NaPi4 phosphate transporter is observed by immunofluorescence in the apical membrane of OK cells. Notice the decrease in the red signal, and (d) this is confirmed by western blot in isolated OK cell BBM. At least n = 3 per group. BBM, brush border membrane; DMHCA, N,N-dimethyl-3β-hydroxy-cholenamide; NaPi, sodium-phosphate; NS, nonsignificant; OK, opossum kidney; Pi, inorganic phosphate; SCD1, stearoyl-CoA desaturase 1.