Table 1.
Blood and urine parameters from vehicle and sirolimus-treated rats after two and seven days of treatment.
Figure 1.
Effect of sirolimus on renal phosphate excretion.
Trajectories of urinary phosphate/creatinine ratios of sirolimus and vehicle treated rats. Sirolimus caused statistically significant increased urinary phosphate excretion 24 hours after the first sirolimus injection (day 2) until after seven sirolimus injection (day 8, n = 6 for each group).
Figure 2.
Sirolimus has no effect on apical renal phosphate transport.
Effect of Sirolimus treatment for two and seven days on BBM sodium-dependent phosphate uptake in absence and presence of phosphonoformic acid (6 mM, PFA), an inhibitor of SLC34 phosphate cotransporter. Sirolimus did not alter 32Pi-uptake in absence (a) or presence (b) of PFA after two and seven days of sirolimus treatment (n = 6 each group). Incubation of BBMV from untreated rats with 20ng/ml and 100ng/ml sirolimus did not change Na+-dependent phosphate fluxes in the absence (c) or presence (d) of PFA.
Table 2.
Serum values of phosphate regulatory hormones from vehicle and sirolimus-treated rats after two and seven days of treatment.
Figure 3.
Sirolimus has no effect on renal phosphate transporter mRNA abundance.
Results of the semiquantitative RT-qPCR for NaPi-IIa, NaPi-IIc, Pit-2, NHE3 and klotho after two and seven days of sirolimus treatment. mRNA abundance for NaPi-IIa, klotho, Pit2 and NHE3 was not different between groups after two and seven days of sirolimus treatment. NaPi-IIc mRNA abundance was significantly lower in sirolimus treated animals after two and seven days. * p<0,05.
Figure 4.
Sirolimus does not alter renal phosphate transporters.
Sirolimus treatment for two and seven days does not alter protein expression levels of NaPi-IIa, NaPi-IIc, Pit-2, klotho and NHE3 in the brush border membrane. Brush border membranes or total membrane protein were prepared from kidneys of sirolimus and vehicle injected rats (n = 6) and 10 µg of brush border membranes or 35 µg of total membrane protein were loaded per lane for immunoblotting. a Membranes were tested for NaPi-IIa, NaPi-IIc, Pit-2, NHE3, and the αNa+/K+-ATPase subunit and stripped for reprobing with ß-actin to control for loading. b Densitometric analysis of all immunoblots with the appropriate software was performed and bands of the proteins of interest were normalized against ß-actin and the respective vehicle groups. Sirolimus treatment for two and seven days did not change the abundance of NaPi-IIa, NaPi-IIc, Pit-2, klotho and NHE3 compared to vehicle.
Figure 5.
Sirolimus has no effect on localization of renal phosphate transporters.
Effect of sirolimus treatment for two and seven days on NaPi-IIa, NaPi-IIc and Pit-2 localization. NaPi-IIa, NaPi-IIc or Pit2 staining (red) was observed in the BBM of early proximal tubules, and colocalized with β-actin as a marker of the BBM (green) as indicated by the yellow overlay. Nuclei were stained with DAPI (blue). No difference was observed between animals treated with vehicle or sirolimus for either 2 or 7 days (n = 5 per group). Original magnification 630 x.