Effects of phosphate intake on distribution of type II Na/Pi cotransporter mRNA in rat kidney. (1/75)

BACKGROUND: Renal phosphate (Pi) reabsorption is regulated by dietary Pi intake, as well as in other ways. Changes in Pi reabsorption are associated with the modulation of sodium/Pi cotransporter type II (NaPi-2) protein abundance in the brush border membrane (BBM) of proximal tubules (PTs) and of renal NaPi-2 mRNA levels. In this study, we address whether the NaPi-2 protein and NaPi-2 mRNA distribution patterns in the renal cortex vary in parallel with changes of dietary Pi intake. METHODS: We investigated in cryosections of perfusion-fixed rat kidneys by in situ hybridization (ISH) and immunohistochemistry (IHC) the distribution patterns of NaPi-2 mRNA and of NaPi-2 protein one week, two hours, and four hours after changes in dietary Pi intake. RESULTS: NaPi-2 mRNA and NaPi-2 protein were present in PTs exclusively. In rats adapted to one week of high Pi intake, signals for NaPi-2 mRNA and NaPi-2 protein in cortical PTs were weak, except in the convoluted parts of PTs of juxtamedullary nephrons. After one week of low Pi intake, the ISH and IHC signals for NaPi-2 were high in PT segments in all cortical levels. The switch from a chronic high to a low Pi intake within two and four hours induced no increase and a slight increase, respectively, in the NaPi-2 mRNA signal in PTs of midcortical and of superficial nephrons, whereas in the BBM of these nephrons, NaPi-2 protein was markedly up-regulated. Two and four hours after switching from low to high Pi intake, the overall high ISH signal for NaPi-2 mRNA was unchanged, whereas NaPi-2 protein staining was drastically down-regulated in the BBM of PTs from superficial and midcortical nephrons. CONCLUSIONS: The marked changes in NaPi-2 protein abundance in the BBM, following altered dietary Pi intake, precede corresponding changes at the RNA level by several hours. Thus, the early adaptation to altered Pi intake involves mRNA-independent mechanisms. The up- or down-regulation of NaPi-2 protein abundance in the BBM and NaPi-2 mRNA in PT affects mainly midcortical and superficial nephrons.  (+info)

Developmental expression of sodium entry pathways in rat nephron. (2/75)

During the past several years, sites of expression of ion transport proteins in tubules from adult kidneys have been described and correlated with functional properties. Less information is available concerning sites of expression during tubule morphogenesis, although such expression patterns may be crucial to renal development. In the current studies, patterns of renal axial differentiation were defined by mapping the expression of sodium transport pathways during nephrogenesis in the rat. Combined in situ hybridization and immunohistochemistry were used to localize the Na-Pi cotransporter type 2 (NaPi2), the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), the thiazide-sensitive Na-Cl cotransporter (NCC), the Na/Ca exchanger (NaCa), the epithelial sodium channel (rENaC), and 11beta-hydroxysteroid dehydrogenase (11HSD). The onset of expression of these proteins began in post-S-shape stages. NKCC2 was initially expressed at the macula densa region and later extended into the nascent ascending limb of the loop of Henle (TAL), whereas differentiation of the proximal tubular part of the loop of Henle showed a comparatively retarded onset when probed for NaPi2. The NCC was initially found at the distal end of the nascent distal convoluted tubule (DCT) and later extended toward the junction with the TAL. After a period of changing proportions, subsegmentation of the DCT into a proximal part expressing NCC alone and a distal part expressing NCC together with NaCa was evident. Strong coexpression of rENaC and 11HSD was observed in early nascent connecting tubule (CNT) and collecting ducts and later also in the distal portion of the DCT. Ontogeny of the expression of NCC, NaCa, 11HSD, and rENaC in the late distal convolutions indicates a heterogenous origin of the CNT. These data present a detailed analysis of the relations between the anatomic differentiation of the developing renal tubule and the expression of tubular transport proteins.  (+info)

Modulation of phosphate uptake and amphotropic murine leukemia virus entry by posttranslational modifications of PIT-2. (3/75)

PIT-2 is a type III sodium phosphate cotransporter and the receptor for amphotropic murine leukemia viruses. We have investigated the expression and the functions of a tagged version of PIT-2 in CHO cells. PIT-2 remained equally abundant at the cell surface within 6 h following variation of the phosphate supply. In contrast, the efficiency of phosphate uptake and retrovirus entry was inversely related to the extracellular phosphate concentration, indicating that PIT-2 activities are modulated by posttranslational modifications of cell surface molecules induced by phosphate. Conformational changes of PIT-2 contribute to both activities, as shown by the inhibitory effect of sulfhydryl reagents known as inhibitors of type II cotransporters. A physical association of PIT-2 with actin was demonstrated. Modifications of the actin network were induced by variations of the concentrations of extracellular phosphate, cytochalasin D, or lysophosphatidic acid. They revealed that the formation of actin stress fibers determines the cell surface distribution of PIT-2, the internalization of the receptor in response to virus binding, and the capacity to process retrovirus entry. Thus, the presence of PIT-2 at the cell surface is not sufficient to ensure phosphate transport and susceptibility to amphotropic retrovirus infection. Further activation of cell surface PIT-2 molecules is required for these functions.  (+info)

Stoichiometry and Na+ binding cooperativity of rat and flounder renal type II Na+-Pi cotransporters. (4/75)

The stoichiometry of the rat and flounder isoforms of the renal type II sodium-phosphate (Na+-Pi) cotransporter was determined directly by simultaneous measurements of phosphate (Pi)-induced inward current and uptake of radiolabeled Pi and Na+ in Xenopus laevis oocytes expressing the cotransporters. There was a direct correlation between the Pi-induced inward charge and Pi uptake into the oocytes; the slope indicated that one net inward charge was transported per Pi. There was also a direct correlation between the Pi-induced inward charge and Na+ influx; the slope indicated that the influx of three Na+ ions resulted in one net inward charge. This behavior was similar for both isoforms. We conclude that for both Na+-Pi cotransporter isoforms the Na+:Pi stoichiometry is 3:1 and that divalent Pi is the transported substrate. Steady-state activation of the currents showed that the Hill coefficients for Pi were unity for both isoforms, whereas for Na+, they were 1.8 (flounder) and 2.5 (rat). Therefore, despite significant differences in the apparent Na+ binding cooperativity, the estimated Na+:Pi stoichiometry was the same for both isoforms.  (+info)

cAMP-dependent and -independent downregulation of type II Na-Pi cotransporters by PTH. (5/75)

Parathyroid hormone (PTH) leads to the inhibition of Na-Pi cotransport activity and to the downregulation of the number of type II Na-Pi cotransporters in proximal tubules, as well as in opossum kidney (OK) cells. PTH is known also to lead to an activation of adenylate cyclase and phospholipase C in proximal tubular preparations, as well as in OK cells. In the present study, we investigated the involvement of these two regulatory pathways in OK cells in the PTH-dependent downregulation of the number of type II Na-Pi cotransporters. We have addressed this issue by using pharmacological activators of protein kinase A (PKA) and protein kinase C (PKC), i.e., 8-bromo-cAMP (8-BrcAMP) and beta-12-O-tetradecanoylphorbol 13-acetate (beta-TPA), respectively, as well as by the use of synthetic peptide fragments of PTH that activate adenylate cyclase and/or phospholipase C, i.e., PTH-(1-34) and PTH-(3-34), respectively. Our results show that PTH signal transduction via cAMP-dependent, as well as cAMP-independent, pathways leads to a membrane retrieval and degradation of type II Na-Pi cotransporters and, thereby, to the inhibition of Na-Pi cotransport activity. Thereby, the cAMP-independent regulatory pathway leads only to partial effects (approximately 50%).  (+info)

Protein kinase C activators induce membrane retrieval of type II Na+-phosphate cotransporters expressed in Xenopus oocytes. (6/75)

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 4alpha-PDD (4alpha-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.  (+info)

Distribution of the sodium/phosphate transporter during postnatal ontogeny of the rat kidney. (7/75)

Renal phosphate reabsorption via the type II sodium/ phosphate cotransporter (NaPi-2) in the brush border membrane (BBM) of proximal tubules underlies alterations during aging. The ontogeny of NaPi-2 in kidneys from newborn to 6-wk-old rats was investigated. NaPi-2 protein distribution in the kidneys of neonatal, 13-d-old, 22-d-old, and 6-wk-old rats was immunohistochemically analyzed, and NaPi-2 mRNA distribution in neonatal and 6-wk-old rats was analyzed by in situ hybridization. In kidneys of newborn rats, the appearance of NaPi-2 protein and mRNA coincided with the development of the brush border (assessed by actin staining) on proximal tubular cells. NaPi-2 was not detectable in the nephrogenic zone or in the outgrowing straight sections of proximal tubules, which lack a brush border. In 13-d-old suckling rats, strong NaPi-2 staining was seen in the BBM of convoluted proximal tubules of all nephron generations. In contrast, in 22-d-old weaned rats, NaPi-2 staining in the BBM of superficial nephrons was weaker than that in the BBM of juxtamedullary nephrons. Western blotting demonstrated that the overall abundance of NaPi-2 protein in the BBM of 22-d-old rats was decreased to approximately 70% of that in 13-d-old rats. In kidneys of 6-wk-old rats, the internephron gradient for NaPi-2 abundance in the BBM corresponded to that in adult rats. The data suggest that the NaPi-2 system in the kidney is fully functional and possesses the capacity for regulation as soon as nephrogenesis is completed. The manifestation of NaPi-2 internephron heterogeneity immediately after weaning might be related to the change in dietary inorganic phosphate content.  (+info)

Kidney cortex cells derived from SV40 transgenic mice retain intrinsic properties of polarized proximal tubule cells. (8/75)

BACKGROUND: We have developed a nontransformed immortalized mice kidney cortex epithelial cell (MKCC) culture from a mouse transgenic for a recombinant plasmid adeno-SV40 (PK4). Methods and Results. After 12 months in culture, the immortalized cells had a stable homogeneous epithelial-like phenotype, expressed simian virus 40 (SV40) T-antigen, but failed to induce tumors after injection in nude mice. Epithelium exhibited polarity with an apical domain bearing many microvilli separated from lateral domains by junctional complexes with ZO1 protein. The transepithelial resistance was low. A Na-dependent glucose uptake sensitive to phlorizin and a Na-dependent phosphate uptake sensitive to arsenate were present. Western blot analysis of membrane fractions showed that anti-Na-Pi antiserum reacted with a 87 kD protein. The Na/H antiporters NHE-1, NHE-2, and NHE-3 mRNAs were detected by reverse transcription-polymerase chain reaction (RT-PCR). The corresponding proteins with molecular weights of 111, 81, and 75 kD, respectively, could be detected by Western blot and were shown to be functional. Parathyroid hormone (PTH) induced a tenfold increase in cAMP and reduced the Na-dependent phosphate uptake and NHE-3 activity, as observed in proximal tubule cells. Isoforms alpha, delta, epsilon, and zeta of protein kinase C (PKC) were present in the cells. Angiotensin II (Ang II) elicited a translocation of the PKC-alpha toward the basolateral and apical domains. CONCLUSION: Thus, the MKCC culture retains the structural and functional properties of proximal tubular cells. To our knowledge, it is the first cell culture obtained from transgenic mice that exhibits the NHE-3 antiporter and type II Na-Pi cotransporter. MKCCs also display functional receptors for PTH and Ang II. Thus, MKCCs offer a powerful in vitro system to study the cellular mechanisms of ion transport regulation in proximal epithelium.  (+info)