Relative contributions of Na+-dependent phosphate co-transporters to phosphate transport in mouse kidney: RNase H-mediated hybrid depletion analysis

Reabsorption of P-i in the proximal tubule of the kidney is an important determinant of P-i homoeostasis. At least three types (types I-III) of high-affinity Na+-dependent P-i co-transporters have been identified in mammalian kidneys. The relative roles of these three types of Na+/P-i co-transporters in P-i transport in mouse kidney cortex have now been investigated by RNase H-mediated hybrid depletion. Whereas isolated brush-border membrane vesicles showed the presence of two kinetically distinct Na+/P-i co-transport systems (high K-m-low V-max and low K-m-high V-max), Xenopus oocytes, microinjected with polyadenylated [poly(A)(+)] RNA from mouse kidney cortex, showed only the high-affinity P-i uptake system. Kidney poly(A)(+) RNA was incubated in vitro with antisense oligonucleotides corresponding to Npt-1 (type I), NaPi-7 (type II) or Glvr-1 (type III) Na+/P-i co-transporter mRNAs, and then with RNase H. Injection of such treated RNA preparations into Xenopus oocytes revealed that an NaPi-7 antisense oligonucleotide that resulted in complete degradation of NaPi-7 mRNA (as revealed by Northern blot analysis), also induced complete inhibition of P-i uptake. Degradation of Npt-1 or Glvr-1 mRNAs induced by corresponding antisense oligonucleotides had no effect on P-i transport, which was subsequently measured in oocytes. These results indicate that the type II Na+/P-i co-transporter NaPi-7 mediated most Na+-dependent P-i transport in mouse kidney cortex.