IGF-I and vanadate stimulate Na/Pi-cotransport in OK cells by increasing type II Na/Pi-cotransporter protein stability

 Insulin-like growth factor (IGF)-I and vanadate increase Na-dependent phosphate (Na/P_i) cotransport in opossum kidney (OK) cells. To gain more information about the mechanisms by which IGF-I and vanadate stimulate Na/P_i-cotransport, we measured type II Na/P_i-cotransporter (NaPi-4) protein abundance by Western blot analysis and investigated the effects of protein synthesis and tyrosine kinase inhibitors. The key findings in the present studies are as follows. First, incubation in IGF-I (10^–8 M) and/or vanadate (10^–3 M) for 3 h led to a non-additive 1.4-fold increase in Na/P_i-cotransport activity which was paralleled by a 1.5- to 2-fold increase in NaPi-4 protein. Second, actinomycin D did not abolish the increase in Na/P_i-cotransport and cycloheximide did not prevent the IGF-I-induced increase in Na/P_i-cotransport and NaPi-4 protein. Third, among the protein kinase inhibitors tested, only staurosporine substantially reduced the stimulation of Na/P_i-cotransport. In conclusion, the stimulatory effect of IGF-I on Na/P_i-cotransport is paralleled by an increased expression of NaPi-4 protein that is independent of protein synthesis and therefore results from increased protein stability. The observation that IGF-I and/or vanadate lead to similar increases in Na/P_i-cotransport and NaPi-4 protein abundance provides further evidence that the stimulation of Na/P_i-cotransport by IGF-I and vanadate involves protein tyrosine phosphorylation of the same signalling molecules. Received: 1 May 1998 / Received after revision: 25 August 1998 / Accepted: 1 September 1998     

Renal expression of Na+-phosphate cotransporter mRNA and protein: Effect of the Gy mutation and low phosphate diet

The X-linked Gy mutation is closely linked, but not allelic, to Hyp and is characterized by rickets, hypophosphatemia, decreased renal tubular maximum for phosphate (Pi) reabsorption (Tm_P) and a specific reduction in renal brush-border membrane (BBM) Na⁺-Pi cotransport. Gy mice, like their normal littermates, respond to a low-Pi diet with an increase in BBM Na⁺-Pi cotransport, but fail to show an adaptive increase in Tm_p. Using an antibody raised against the NH₂ terminal peptide of the rat renal-specific Na⁺-Pi cotransporter (NaPi-2) and a NaPi-2 cDNA probe, we examined the effect of the Gy mutation and low-Pi diet (0.03% Pi) on NaPi-2 protein and mRNA abundance. The reduction in BBM Na⁺-Pi cotransport in Gy mice (51 ± 5% of normal, P < 0.05) was associated with a decrease in NaPi-2 protein (46 ± 12% of normal, P < 0.05) and mRNA abundance (76 ± 5%, P < 0.05). The low-Pi diet elicited a two- to three-fold increase in Na⁺-Pi cotransport in both normal and Gy mice that was accompanied by a large increase in NaPi-2 protein (10.2-fold in normal and 16.9-fold in Gy mice) and a modest increase in NaPi-2 mRNA (1.3-fold in both mouse strains, P < 0.05). The present data demonstrate that (1) the renal defect in BBM Pi transport in Gy mice can be ascribed to a deficit in NaPi-2 protein and mRNA abundance, (2) both normal and Gy mice respond to low Pi with an adaptive increase in NaPi-2 protein that exceeds the increase in Na⁺-Pi cotransport activity and NaPi-2 mRNA, (3) the adaptive increase in NaPi-2 protein and mRNA are not sufficient for the overall increase in Tm_P following Pi restriction. Received: 27 October 1995 / Received after revision: 4 December 1995 / Accepted: 6 December 1995     

A molecular view of proximal tubular inorganic phosphate (Pi) reabsorption and of its regulation

 In recent years, two mammalian proximal tubular brush border membrane Na/P_i cotransporters (types I and II) have been structurally identified by expression cloning techniques. Oocyte expression studies have shown that only the transport characteristics of the type II transporter correspond to the well-known properties of proximal tubular brush border membrane of P_i transport. In studies on physiological regulation by hormonal and non-hormonal factors a direct involvement and determining role of the type II transporter has been documented. Most interestingly, specific membrane retrieval/insertion phenomena participate in acute (minutes/hours) adjustments of brush border membrane Na/P_i cotransport rates; for chronic (hours/days) alterations also specific resynthesis/degradation processes participate. In pathophysiological alterations (e.g. in X-linked hypophosphataemia and in heavy metal-induced nephrotoxicity) the expression of the type II Na/P_i cotransporters is reduced and explains the observed phosphaturia.     

The sodium phosphate cotransporter family SLC34

This review summarizes the characteristics of the solute carrier family SLC34 that is represented by the type ll Na/P(i)-cotransporters NaPi-lla (SLC34A1), NaPi-llb (SLC34A2) and NaPi-llc (SLC34A3). Other Na/P(i)-cotransporters are described within the SLC17 and SLC20 families. Type ll Na/P(i)-cotransporters are expressed in several tissues and play a major role in the homeostasis of inorganic phosphate. In kidney and small intestine, type ll Na/P(i)-cotransporters are located at the apical sites of epithelial cells and represent the rate limiting steps for transepithelial movement of phosphate. Physiological and pathophysiological regulation of renal and small intestinal epithelial transport of phosphate occurs through alterations in the abundance of type ll Na/P(i)-cotransporters.     

Effects of phosphate intake on distribution of type II Na/Pi cotransporter mRNA in rat kidney

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.