Impaired PTH-induced endocytotic down-regulation of the renal type IIa Na+/Pi-cotransporter in RAP-deficient mice with reduced megalin expression

Abstract

Inorganic phosphate (P i ) reabsorption in the renal proximal tubule occurs mostly via the Na + /P i cotransporter type IIa (NaP i -IIa) located in the brushborder membrane (BBM) and is regulated, among other factors, by dietary P i intake and parathyroid hormone (PTH). The PTH-induced inhibition of P i reabsorption is mediated by endocytosis of Na/P i -IIa from the BBM and subsequent lysosomal degradation. Megalin is involved in receptor-mediated endocytosis of proteins from the urine in the renal proximal tubule. The recently identified receptor-associated protein (RAP) is a novel type of chaperone responsible for the intracellular transport of endocytotic receptors such as megalin. Gene disruption of RAP leads to a decrease of megalin in the BBM and to a disturbed proximal tubular endocytotic machinery. Here we investigated whether the distribution of NaP i -IIa and/ or its regulation by dietary P i intake and PTH is affected in the proximal tubules of RAP-deficient mice as a model for megalin loss. In RAP-deficient mice megalin expression was strongly reduced and restricted to a subapical localization. NaP i -IIa protein distribution and abundance in the kidney was not altered. The localization and abundance of the NaP i -IIa interacting proteins MAP17, PDZK-1, D-AKAP2, and NHE-RF1 were also normal. Other transport proteins expressed in the BBM such as the Na + /H + exchanger NHE-3 and the Na + /sulphate cotransporter NaSi were normally expressed. In whole animals and in isolated fresh kidney slices the PTH-induced internalization of NaP i -IIa was strongly delayed in RAPdeficient mice. PTH receptor expression in the proximal tubule was not affected by the RAP knock-out. cAMP, cGMP or PKC activators induced internalization which was delayed in RAP-deficient mice. In contrast, both wildtype and RAP-deficient mice were able to adapt to high-, normal, and low-P i diets appropriately as indicated by urinary P i excretion and NaP i -IIa protein abundance.

Acute parathyroid hormone differentially regulates renal brush border membrane phosphate cotransporters

“…We and others have shown that PTH causes phosphate excretion at least in part by inducing the internalization of the NaPi-IIa phosphate cotransporter from the brush border membrane of proximal tubules in rats and mice (2,3,7,12,30,48). NaPi-IIa is then routed via clathrin-coated pits and early endosomes to lysosomes and subsequently undergoes degradation (3,21,34,39).…”

Kidney-Specific Inactivation of the Megalin Gene Impairs Trafficking of Renal Inorganic Sodium Phosphate Cotransporter (NaPi-IIa)

“…For slice preparation, kidneys from untreated Cre(ϩ) and Cre(Ϫ) mice were prepared and slices incubated as described (16). Onemillimeter-thick kidney slices were left untreated for control, or treated with PTH (1-34) fragment, 8-Br-cAMP, 8-Br-cGMP, or 1,2-dioctanoyl-sn-glycerol (DOG).…”

“…For HRP histochemistry, cryostat sections were incubated with diaminobenzidine added with H 2 O 2 . Controlling for general BBM antibody staining intensity we have tested antibodies against sodium sulfate cotransporter (NaSi-1) and sodium hydrogen exchanger-3 (NHE-3) as described (16).…”

“…After incubation, slices were transferred to fixative (16), mounted onto cork plates, and frozen. Cryostat sections were then incubated with NaPi-IIa antibody (dilution 1:1500) followed by swine antirabbit IgG conjugated to FITC (Dako).…”

“…The role of receptor-associated protein (RAP), which has been identified as a chaperone-like protein interfering with megalin and related receptors, has further been discussed in this context. The RAP knockout mouse showed a retardation in PTHinduced effect that may be related to an altered distribution of megalin (15,16). An interaction of megalin with a membrane protein of the proximal tubule has further been reported (17).…”