Targeted disruption of the mouse NHERF-1 gene promotes internalization of proximal tubule sodium-phosphate cotransporter type IIa and renal phosphate wasting

Abstract

Na+/H+ exchanger regulatory factor (NHERF)-1 and NHERF-2, two structurally related protein adapters containing tandem PSD-95/Discs large/ZO-1 (PDZ) domains, were identified as essential factors for protein kinase A-mediated inhibition of the sodium-hydrogen exchanger, NHE3. NHERF-1 and NHERF-2 also bound other cellular targets including the sodium-phosphate cotransporter type IIa encoded by the NPT2 gene. Targeted disruption of the mouse NHERF-1 gene eliminated NHERF-1 expression in kidney and other tissues of the mutant mice without altering NHERF-2 levels in these tissues. NHERF-1 (+/-) and (-/-) male mice maintained normal blood electrolytes but showed increased urinary excretion of phosphate when compared with wild-type (+/+) animals. Although the overall levels of renal NHERF-1 targets, NHE3 and Npt2, were unchanged in the mutant mice, immunocytochemistry showed that the Npt2 protein was aberrantly localized at internal sites in the renal proximal tubule cells. The mislocalization of Npt2 paralleled a reduction in the transporter protein in renal brush-border membranes isolated from the mutant mice. In contrast, NHE3 was appropriately localized at the apical surface of proximal tubules in both wild-type and mutant mice. These data suggested that NHERF-1 played a unique role in the apical targeting and/or trafficking of Npt2 in the mammalian kidney, a function not shared by NHERF-2 or other renal PDZ proteins. Phosphate wasting seen in the NHERF-1(-/-) null mice provided a new experimental system for defining the role of PDZ adapters in the hormonal control of ion transport and renal disease.

Fig 1.

Targeted mutation of mouse NHERF-1 gene. (A) The NotI genomic fragments, A5 and B12, from the mouse NHERF-1 gene were used to design a targeting vector. PCR product encompassing the BstII/XbaI fragment from A5 and the NotI/KpnI fragment from B12 were ligated to Neo^r gene, which was combined with herpes simplex virus thymidylate kinase (HSV-TK; KpnI fragment) to construct a targeting vector. Primers based on the shared BstII/XbaI fragment (primer 1) and the Neo^r gene (primer 2) or the first exon of the mouse NHERF-1 gene (primer 3) were used to amplify the WT and mutant alleles by using the PCR. B, BstII; X, XbaI; K, KpnI; N, NotI; S, SacI (indicate the restriction sites). (B) Products generated by PCR amplification of genomic DNA from WT (+/+), heterozygous (+/−), and null (−/−) mice. (C) Southern blot of the BglII digest of mouse genomic DNA hybridized with the PCR product from the A5 region described in Materials and Methods.

Fig 2.

Expression of NHERF isoforms in WT and NHERF-1 mutant mice. (A) Immunoblots of kidney extracts from WT (+/+), heterozygous (+/−), and NHERF-1-null (−/−) mice by using the monospecific antibody against full-length NHERF-1 (1:1,000 dilution) and the anti-peptide antibody against NHERF-2 (1:100 dilution). The secondary antibody, anti-rabbit IgG, was used at a 1:5,000 dilution. (B) Immunostaining of renal proximal tubules from WT and NHERF-1-null mice by using the same anti-NHERF-1 and -NHERF-2 antibodies (both at 10 μg/ml protein concentration). (Bar = 100 μm.)

Fig 3.

Expression of NHERF-1 targets in renal tissue from WT and NHERF-1-null mice. (A) The immunoblots of kidney lysates and isolated brush-border membranes (BBM) from WT and NHERF-1-null mice by using antibodies (at 1:1,000 dilution) against Npt2 (L697) and NHE3 (Chemicon). (B) The immunostaining of kidney proximal tubules from WT and NHERF-1-null mice by using the same anti-Npt2 and anti-NHE3 antibodies. (Bar = 100 μm.)