Martin Traebert

Interaction of the Type IIa Na/Pi Cotransporter with PDZ Proteins

The type IIa Na+-dependent inorganic phosphate (Na/Pi) cotransporter is localized in the apical membrane of proximal tubular cells and is regulated by an endocytotic pathway. Because molecular processes such as apical sorting, internalization, or subsequent degradation might be assisted by associated proteins, a yeast two-hybrid screen against the C-terminal, cytosolic tail of type IIa cotransporter was designed. Most of the potential proteins found belonged to proteins with multiple PDZ modules and were either identical/related to PDZK1 or identical to NHERF-1. Yeast trap truncation assays confined the peptide-protein association to the C-terminal amino acid residues TRL of type IIa cotransporter and to single PDZ domains of each identified protein, respectively. The specificity of these interactions were confirmed in yeast by testing other apical localized transmembraneous proteins. Moreover, the type IIa protein was recovered in vitro by glutathioneS-transferase-fused PDZ proteins from isolated renal brush border membranes or from type IIa-expressing oocytes. Further, these PDZ proteins are immunohistochemically detected either in the microvilli or in the subapical compartment of proximal tubular cells. Our results suggest that the type IIa Na/Pi cotransporter interacts with various PDZ proteins that might be responsible for the apical sorting, parathyroid hormone controlled endocytosis or the lysosomal sorting of internalized type IIa cotransporter.

Regulation of small intestinal Na-Pi type IIb cotransporter by dietary phosphate intake

Dietary restriction of phosphate is a well-known stimulator (acting indirectly via vitamin D3) of small intestinal apical Na-Picotransport. In the present study, we document by Western blots and immunohistochemistry that, in mice, a low-Pi diet given for several days leads (in parallel to a stimulation of Na-Pi cotransport) to an increase of the abundance of the type IIb Na-Pi 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-Pi diet or cholecalciferol. It is concluded that stimulation of intestinal Na-Pi cotransport by low-Pi diet and vitamin D3 can be explained by an increased amount of type IIb Na-Picotransporters in the brush-border membrane and that augmentation of type IIb Na-Pi cotransporters is not related to an increased rate of transcription of the type IIb gene.

Posttranscriptional regulation of the proximal tubule NaPi-II transporter in response to PTH and dietary Pi

The rate of proximal tubular reabsorption of phosphate (Pi) is a major determinant of Pi homeostasis. Deviations of the extracellular concentration of Piare corrected by many factors that control the activity of Na-Pi cotransport across the apical membrane. In this review, we describe the regulation of proximal tubule Pi reabsorption via one particular Na-Pi 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 Pi and is considered irreversible since internalized type IIa Na-Picotransporters are subsequently routed to the lysosomes for degradation.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.

Studies on the topology of the renal type II NaPi-cotransporter.

Abstract The rat type II sodium/phosphate cotransporter (NaPi-2) is a 85- to 90-kDa glycosylated protein located at the proximal tubular brush border membrane. Hydropathy predictions suggest eight transmembrane domains (sTM) with a large glycosylated loop between sTM 3 and sTM 4. We have studied the membrane topology of NaPi-2 expressed in oocytes. A 33-amino-acid fragment containing the FLAG epitope was inserted into seven loops connecting the sTMs and into the NH2- and COOH-ends of the protein. FLAG-antibody binding suggested that the loops connecting sTM 1 and sTM 2 as well as sTM 3 and sTM 4 are located extracellularly. Based on the lack of FLAG-antibody binding we suggest intracellular locations for the NH2- and COOH-termini and the region connecting sTM 4 and sTM 5. Immunoprecipitation studies of in vitro translated protein also suggest that the NH2-terminus is sited extracellularly. In immunohistochemical studies with NaPi-2-transfected MDCK cells, an interaction with NH2- and COOH- terminal antipeptide antibodies could only be obtained after membrane permeabilization. The presented data are an experimental documentation of the intracellular location of the NH2- and COOH-termini, and of the extracellular location of extracellular loops 1 and 2.

Expression of type II Na-Picotransporter in alveolar type II cells

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.Type II Na-Pi cotransporters (type IIa and type IIb) represent apically located Na-Pi 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-Pi 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-Pidiet. After a chronic adaptation to a low-Pi 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-Pi cotransporter in alveolar type II cells.

Dietary phosphate and parathyroid hormone alter the expression of the calcium-sensing receptor (CaR) and the Na+-dependent Pi transporter (NaPi-2) in the rat proximal tubule.

Abstract. Dietary phosphate (Pi) intake and parathyroid hormone (PTH) are essential regulators of proximal tubular (PT) Pi reabsorption; both factors are associated with adaptive changes in PT apical brush border membrane (BBM) Na/Pi-cotransport activity and specific transporter protein (NaPi-2) content. Urinary Pi excretion is also inversely correlated with luminal Ca2+ concentration ([Ca2+]) both in a PTH-dependent and -independent fashion. A cell-surface, Ca2+(/polyvalent cation)-sensing receptor (CaR) has been localized to the PT BBM with unknown function. To investigate whether PTH and/or dietary Pi intake could affect the distribution or the expression of the CaR, we evaluated their effects on rat kidney CaR and the NaPi-2 expression by Western blot analysis and immunofluorescence microscopy. A chronic high-Pi (1.2%) versus low-Pi (0.1%) diet and acute PTH (1–34) infusion significantly reduced the PT BBM expression of both NaPi-2 and CaR proteins. CaR-specific immunoreactivity in nephron segments other than the PT was not affected by PTH or Pi intake. These results suggest that reduced renal PT CaR expression by a high-Pi diet and by increased circulating PTH levels could contribute to the local control of PT handling of Ca2+ and Pi.

Protein kinase C activators induce membrane retrieval of type II Na+‐phosphate cotransporters expressed in Xenopus oocytes

1 The rate of inorganic phosphate (Pi) reabsorption in the mammalian kidney is determined by the amount of type II sodium‐coupled inorganic phosphate (Na+‐Pi) cotransport protein present in the brush border membrane. Under physiological conditions, parathyroid hormone (PTH) leads to an inhibition of Na+‐Pi cotransport activity, most probably mediated by the protein kinase A (PKA) and/or C (PKC) pathways. 2 In this study, PKC‐induced inhibition of type II Na+‐Pi cotransport activity was characterized in Xenopus laevis oocytes using electrophysiological and immunodetection techniques. Transport function was quantified in terms of Pi‐activated current. 3 Oocytes expressing the type IIa rat renal, type IIb flounder renal or type IIb mouse intestinal Na+‐Pi cotransporters lost > 50% of Pi‐activated transport function when exposed to the PKC activators DOG (1,2‐dioctanoyl‐sn‐glycerol) or PMA (phorbol 12‐myristate 13‐acetate). DOG‐induced inhibition was partially reduced with the PKC inhibitors staurosporine and bisindolylmaleimide I. Oocytes exposed to the inactive phorbol ester 4α‐PDD (4α‐phorbol 12,13‐didecanoate) showed no significant loss of cotransporter function. 4 Oocytes expressing the rat renal Na+‐SO42‐ cotransporter alone, or coexpressing this with the type IIa rat renal Na+‐Pi cotransporter, showed no downregulation of SO42‐‐activated cotransport activity by DOG. 5 Steady‐state and presteady‐state voltage‐dependent kinetics of type II Na+‐Pi cotransporter function were unaffected by DOG. 6 DOG induced a decrease in membrane capacitance which indicated a reduction in membrane area, thereby providing evidence for PKC‐mediated endocytosis. 7 Immunocytochemical studies showed a redistribution of type II Na+‐Pi cotransporters from the oolemma to the submembrane region after DOG treatment. Surface biotinylation confirmed a DOG‐induced internalization of the transport protein. 8 These findings document a specific retrieval of exogenous type II Na+‐Pi cotransporters induced by activation of a PKC pathway in the Xenopus oocyte.

Regulation of the renal type IIa Na/Pi cotransporter by cGMP

Inhibition of proximal tubular phosphate (Pi) reabsorption involves, as far as we know, brush border membrane retrieval of the type IIa Na/Pi-cotransporter. The aim of the present study was to analyze whether intracellular cGMP-mediated regulation of Pi reabsorption also involves retrieval of the type IIa Na/Pi-cotransporter, as previously shown for cAMP. Atrial natriuretic peptide (ANP) and nitric oxide (NO) were used to stimulate guanylate cyclase. In vivo perfusion of mice kidneys with either ANP or NO donors resulted in a downregulation of type IIa Na/Pi-cotransporters on the brush border membranes of proximal tubules. These effects were mimicked by activation of protein kinase G with 8Br-cGMP. In in-vitro-perfused mice proximal tubules, ANP was effective when added either to the apical or basolateral perfusate, suggesting the presence of receptors on both membrane sites. The effects of ANP and NO were blocked by the protein kinase G inhibitor LY 83553. Parallel experiments in OK cells, a renal proximal tubule model, provided similar information. Our findings document that cGMP-mediated regulation (ANP and NO) of type IIa Na/Pi-cotransporters also takes place via internalization of the transporter protein.

An apical membrane Na+/H+ exchanger isoform, NHE-3, is present in the rat epididymal epithelium

Abstract. An acidic milieu is required for sperm maturation and for keeping sperm quiescent during storage in the cauda epididymidis. Previous studies have implicated a Na+/H+ exchanger (NHE) in epididymal acidification together with carbonic anhydrase (CA) and vacuolar proton adenosine triphosphatase (H+-ATPase). The present studies were undertaken to discover whether the NHE isoform involved is NHE-3, which is known to mediate Na+ and HCO3– absorption in renal tubules. Using the reverse transcription polymerase chain reaction technique (RT-PCR), Northern blot analysis and in situ hybridization, NHE-3 mRNA was detected mainly in the cauda epididymis and to a lesser extent in other regions of the epididymis. Immunohistochemical studies showed that NHE-3 was present in the apical membranes of the epithelial principal cells and confirmed that its expression is strongest in the cauda region, decreasing towards the more proximal regions. Immunoblotting showed a similar expression pattern. These results demonstrate that NHE-3 is expressed in the rat epididymal duct with strongest expression in its cauda region. These findings are thus consistent with the possibility that NHE-3 in the epididymal duct is involved in luminal Na+ and/or HCO3– absorption, as in the renal proximal tubule, and thereby in the regulation of sperm motility and maturation.

Cleavage of disulfide bonds leads to inactivation and degradation of the type IIa, but not type IIb sodium phosphate cotransporter expressed in Xenopus laevis oocytes.

Abstract. Tris(2-carboxyethyl)phosphine (TCEP) reduces (cleaves) disulfide bonds of the renal proximal tubule type IIa Na/Pi- cotransporter (rat NaPi IIa) and thereby inhibits its function. We tested the effect of TCEP on the murine type IIa Na/Pi-cotransporter and the corresponding IIb intestinal isoform both expressed in Xenopus laevis oocytes. After incubation with TCEP the function of NaPi IIa was inhibited and protein amount was decreased. Injection of the lysosomal inhibitor leupeptin prevented degradation of the protein. Exposure of oocytes to TCEP at 0°C led to a reduction in transport function without concomitant loss in Na/Pi IIa protein. In contrast to NaPi type IIa, the type IIb isoform was neither inhibited, nor degraded after incubation with TCEP. These results suggest that cleavage of disulfide bonds led to changes within the confirmation of the type IIa transporter that result in (i) inhibition of the transport activity and (ii) internalization and subsequent lysosomal degradation of transporter protein. Sequence comparisons suggest the involvement/presence of different disulfide bonds in type IIa and type IIb Na/Pi-cotransporters.