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Traebert, M., J. Roth, J. Biber, H. Murer, and B. Kaissling. Internalization of proximal tubular type II Na-[P.sub.i] cotransporter by PTH: immunogold electron microscopy. Am. J. Physiol. Renal Physiol. 278: F148-F154, 2000.--Physiological/pathophysiological alterations in proximal tubular [P.sub.i] reabsorption are associated with an altered brush-border membrane (BBM) expression of type II Na-[P.sub.i] cotransporter molecules. Reduction is achieved by an internalization and lysosomal degradation and an increase in [P.sub.i] reabsorption by new synthesis and BBM insertion of type II Na-[P.sub.i] cotransporters. In the present study, we investigated by immunohistochemistry and immunogold electron microscopy the routing of internalized rat type II Na-[P.sub.i] cotransporters (NaPi-2). In kidney of rats on a chronic low-[P.sub.i] diet, NaPi-2 is mainly localized in the BBM, in cisterns of the Golgi apparatus and sparsely also in large endocytotic vacuoles and lysosomes. Fifteen minutes after the injection of the 1-34 analog of parathyroid hormone (PTH), the amount of NaPi-2 was decreased in the BBM and increased in endocytotic vesicles. NaPi-2 molecules colocalized with horseradish peroxidase injected prior to the injection of PTH. Vesicles labeled for NaPi-2 were occasionally also labeled for clathrin or the adaptor protein AP2. We conclude that NaPi-2 molecules enter the subapical compartment from where NaPi-2-containing vesicles are segregated off and directed to the lysosomes. A clathrin-mediated pathway may contribute to the PTH-induced internalization of NaPi-2. NaPi-2; horseradish peroxidase; endocytosis; immunohistochemistry

Parathyroid hormone (PTH) inhibits proximal tubular reabsorption of P(i) by retrieval of type IIa Na-P(i) cotransporters (NaPi-IIa) from the brush-border membrane (BBM). We analyzed by immunohistochemistry whether PTH analogs, signaling through either protein kinase A (PKA) and C (PKC; 1-34 PTH) or only PKC (3-34 PTH), elicit in rat kidney in vivo or in the perfused murine proximal tubule in vitro a retrieval of NaPi-IIa and whether pharmacological agonists or inhibitors of these kinases are able to either mimic or interfere with these PTH effects. Treatment with either 1-34 or 3-34 PTH downregulated NaPi-IIa in rat kidney. In isolated murine proximal tubules 1-34 PTH was effective when added to either the apical or basolateral perfusate, whereas 3-34 PTH acted only via the luminal perfusate. These effects were mimicked by an activation of PKA with 8-bromoadenosine 3',5'-cyclic monophosphate or PKC with 1, 2-dioctanoylglycerol. The luminal action of both PTH peptides was blocked by inhibition of the PKC pathway (calphostin C), whereas the basolateral effect of 1-34 PTH was completely abolished by inhibiting both pathways (H-89 and calphostin C). These results suggest that 1) NaPi-IIa can be internalized by cAMP-dependent and -independent signaling mechanisms; 2) functional PTH receptors are located in both membrane domains; and 3) apical PTH receptors may preferentially initiate the effect through a PKC-dependent mechanism.

The rate of proximal tubular reabsorption of phosphate (P(i)) is a major determinant of P(i) homeostasis. Deviations of the extracellular concentration of P(i) are corrected by many factors that control the activity of Na-P(i) cotransport across the apical membrane. In this review, we describe the regulation of proximal tubule P(i) reabsorption via one particular Na-P(i) cotransporter (the type IIa cotransporter) by parathyroid hormone (PTH) and dietary phosphate intake. Available data indicate that both factors determine the net amount of type IIa protein residing in the apical membrane. The resulting change in transport capacity is a function of both the rate of cotransporter insertion and internalization. The latter process is most likely regulated by PTH and dietary P(i) and is considered irreversible since internalized type IIa Na-P(i) cotransporters are subsequently routed to the lysosomes for degradation.

Type II Na-P(i) cotransporters (type IIa and type IIb) represent apically located Na-P(i) cotransporters in epithelia of proximal tubules (type IIa) and small intestine (type IIb). Here we provide evidence that the type IIb (but not the type IIa) Na-P(i) cotransporter is also expressed in the lung. With the use of immunohistochemistry, location of the type IIb protein was found exclusively in the apical membrane of type II cells of the alveolar epithelium. Such a location of the type IIb cotransporter suggests an involvement in the reuptake of phosphate necessary for the synthesis of surfactant. A possible regulation of the abundance of the type IIb cotransporter in the lung was studied after adaptation of mice to a low-P(i) diet. After a chronic adaptation to a low-P(i) diet, no changes in the type IIb protein and the type IIb transcript were observed. These results exclude dietary intake of phosphate as a regulatory factor of the type IIb Na-P(i) cotransporter in alveolar type II cells.

Dietary restriction of phosphate is a well-known stimulator (acting indirectly via vitamin D(3)) of small intestinal apical Na-P(i) cotransport. In the present study, we document by Western blots and immunohistochemistry that, in mice, a low-P(i) diet given for several days leads (in parallel to a stimulation of Na-P(i) cotransport) to an increase of the abundance of the type IIb Na-P(i) cotransporter in the brush-border membrane of mouse enterocytes. Similar results were also obtained by an injection of cholecalciferol. The abundance of the type IIb transcript was investigated by Northern blots. These results indicated that the amount of the type IIb transcript was not changed by either low-P(i) diet or cholecalciferol. It is concluded that stimulation of intestinal Na-P(i) cotransport by low-P(i) diet and vitamin D(3) can be explained by an increased amount of type IIb Na-P(i) cotransporters in the brush-border membrane and that augmentation of type IIb Na-P(i) cotransporters is not related to an increased rate of transcription of the type IIb gene.