3 �� 1.2 nmol/mg protein per 10 s in control mice to 17.3 �� 2.8 nmol/mg protein per 10 s in TNBS mice (n = 3; P = 0.028) (Fig. 1A). Western blot with BBMV protein showed that TNBS administration also decreased NaPi-IIb immunoreactive protein abundance (indicated by the ratio of optical densities of the NaPi-IIb band enough to that of the ��-actin band) from 1.97 �� 0.45 in control mice to 0.73 �� 0.18 in TNBS mice (n = 3; P = 0.017) (Fig. 1B). Fig. 1. Effect of inflammation on intestinal phosphate absorption and type IIb sodium-phosphate cotransporter (NaPi-IIb) expression in trinitrobenzene sulfonic acid (TNBS) colitis mice. Six-week-old male mice were treated with TNBS (2 mg/mouse) and were harvested … Effect of TNBS colitis on NaPi-IIb expression in rat intestine.
Male rats received TNBS (1 mg/rat in 50% ethanol) or PBS buffer in a total volume of 250 ��l by an enema into the colonic lumen. Six days after TNBS administration, rats were killed and jejunal mucosa was harvested for BBMV and RNA purification. Western blot was used to determine NaPi-IIb protein abundance from BBMV protein. Real-time PCR was used to determine NaPi-IIb mRNA expression. Western blot results showed that NaPi-IIb immunoreactive protein abundance was reduced by ~53% in TNBS colitis rats, from 1.23 �� 0.11 in control rats to 0.59 �� 0.26 in TNBS rats (n = 3; P = 0.04) (Fig. 2A). Real-time PCR data indicated that the expression of NaPi-IIb mRNA was decreased by ~42% in TNBS colitis rats compared with control rats, from 1.09 �� 0.02 in control rats to 0.63 �� 0.07 in TNBS rats (n = 3; P = 0.004) (Fig.
2B). Fig. 2. Effect of inflammation on NaPi-IIb expression in TNBS colitis rats. Three-week-old rats were treated with TNBS (1 mg/rat) and were harvested 6 days after TNBS administration. A: BBMVs were isolated from the jejunal mucosa of rats treated with PBS or TNBS … Effect of TNF-�� on phosphate uptake and NaPi-IIb expression in Caco-2 cells. The endogenous NaPi-IIb expression in Caco-2 cells has been shown in our previous work (38). To study the effect of TNF-�� on cellular phosphate absorption, Caco-2 cells were grown in 24-well plates and treated with normal or TNF-��-containing medium (20 ng/ml) for 40 h. Cellular phosphate uptake assays were performed to measure the rate of phosphate uptake in Caco-2 cells. Western blot detection was used to assess NaPi-IIb protein levels in Caco-2 cells. Real-time PCR was conducted to determine the expression of NaPi-IIb mRNA in Caco-2 cells. As shown in Fig. 3, sodium-dependent phosphate absorption was significantly decreased in Caco-2 cells after TNF-�� treatment, from 2.51 �� 0.27 nmol/mg protein per 15 min in control cells Brefeldin_A to 1.14 �� 0.37 nmol/mg protein per 15 min in TNF-��-treated cells (n = 3; P = 0.013) (Fig. 3A).