The sodium phosphate cotransporter family and nicotinamide phosphoribosyltransferase contribute to the daily oscillation of plasma inorganic phosphate concentration
2018; Elsevier BV; Volume: 93; Issue: 5 Linguagem: Inglês
10.1016/j.kint.2017.11.022
ISSN1523-1755
AutoresAtsumi Miyagawa, Sawako Tatsumi, Wako Takahama, Osamu Fujii, Kenta Nagamoto, Emi Kinoshita, Kengo Nomura, Kayo Ikuta, Toru Fujii, Ai Hanazaki, Ichiro Kaneko, Hiroko Segawa, Ken–ichi Miyamoto,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoCirculating inorganic phosphate exhibits a remarkable daily oscillation based on food intake. In humans and rodents, the daily oscillation in response to food intake may be coordinated to control the intestinal absorption, renal excretion, cellular shifts, and extracellular concentration of inorganic phosphate. However, mechanisms regulating the resulting oscillation are unknown. Here we investigated the roles of the sodium phosphate cotransporter SLC34 (Npt2) family and nicotinamide phosphoribosyltransferase (Nampt) in the daily oscillation of plasma inorganic phosphate levels. First, it is roughly linked to urinary inorganic phosphate excretion. Second, expression of renal Npt2a and Npt2c, and intestinal Npt2b proteins also exhibit a dynamic daily oscillation. Analyses of Npt2a, Npt2b, and Npt2c knockout mice revealed the importance of renal inorganic phosphate reabsorption and cellular inorganic phosphate shifts in the daily oscillation. Third, experiments in which nicotinamide and a specific Nampt inhibitor (FK866) were administered in the active and rest phases revealed that the Nampt/NAD+ system is involved in renal inorganic phosphate excretion. Additionally, for cellular shifts, liver-specific Nampt deletion disturbed the daily oscillation of plasma phosphate during the rest but not the active phase. In systemic Nampt+/− mice, NAD levels were significantly reduced in the liver, kidney, and intestine, and the daily oscillation (active and rest phases) of the plasma phosphate concentration was attenuated. Thus, the Nampt/NAD+ system for Npt2 regulation and cellular shifts to tissues such as the liver play an important role in generating daily oscillation of plasma inorganic phosphate levels. Circulating inorganic phosphate exhibits a remarkable daily oscillation based on food intake. In humans and rodents, the daily oscillation in response to food intake may be coordinated to control the intestinal absorption, renal excretion, cellular shifts, and extracellular concentration of inorganic phosphate. However, mechanisms regulating the resulting oscillation are unknown. Here we investigated the roles of the sodium phosphate cotransporter SLC34 (Npt2) family and nicotinamide phosphoribosyltransferase (Nampt) in the daily oscillation of plasma inorganic phosphate levels. First, it is roughly linked to urinary inorganic phosphate excretion. Second, expression of renal Npt2a and Npt2c, and intestinal Npt2b proteins also exhibit a dynamic daily oscillation. Analyses of Npt2a, Npt2b, and Npt2c knockout mice revealed the importance of renal inorganic phosphate reabsorption and cellular inorganic phosphate shifts in the daily oscillation. Third, experiments in which nicotinamide and a specific Nampt inhibitor (FK866) were administered in the active and rest phases revealed that the Nampt/NAD+ system is involved in renal inorganic phosphate excretion. Additionally, for cellular shifts, liver-specific Nampt deletion disturbed the daily oscillation of plasma phosphate during the rest but not the active phase. In systemic Nampt+/− mice, NAD levels were significantly reduced in the liver, kidney, and intestine, and the daily oscillation (active and rest phases) of the plasma phosphate concentration was attenuated. Thus, the Nampt/NAD+ system for Npt2 regulation and cellular shifts to tissues such as the liver play an important role in generating daily oscillation of plasma inorganic phosphate levels. Hyperphosphatemia is linked to vascular calcification with chronic kidney disease (CKD), and is an independent risk factor for cardiovascular mortality in hemodialysis patients.1Block G.A. Klassen P.S. Lazarus J.M. et al.Mineral metabolism, mortality, and morbidity in maintenance hemodialysis.J Am Soc Nephrol. 2004; 15: 2208-2218Crossref PubMed Scopus (2213) Google Scholar, 2Hruska K.A. Mathew S. Lund R. et al.Hyperphosphatemia of chronic kidney disease.Kidney Int. 2008; 74: 148-157Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 3Miyamoto K. Haito-Sugino S. Kuwahara S. et al.Sodium-dependent phosphate cotransporters: lessons from gene knockout and mutation studies.J Pharm Sci. 2011; 100: 3719-3730Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar Serum inorganic phosphate (Pi), even within the normal range, is associated with cardiovascular events, cardiovascular mortality, and all-cause mortality, and exhibits a daily oscillation in both healthy individuals and patients with CKD.4Palmer S.C. Hayen A. Macaskill P. et al.Serum levels of phosphorus, parathyroid hormone, and calcium and risks of death and cardiovascular disease in individuals with chronic kidney disease: a systematic review and meta-analysis.JAMA. 2011; 305: 1119-1127Crossref PubMed Scopus (535) Google Scholar, 5Dhingra R. Sullivan L.M. Fox C.S. et al.Relations of serum phosphorus and calcium levels to the incidence of cardiovascular disease in the community.Arch Intern Med. 2007; 167: 879-885Crossref PubMed Scopus (676) Google Scholar, 6Sim J.J. Bhandari S.K. Smith N. et al.Phosphorus and risk of renal failure in subjects with normal renal function.Am J Med. 2013; 126: 311-318Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 7O'Seaghdha C.M. Hwang S.J. Muntner P. et al.Serum phosphorus predicts incident chronic kidney disease and end-stage renal disease.Nephrol Dial Transplant. 2011; 26: 2885-2890Crossref PubMed Scopus (81) Google Scholar, 8Eddington H. Hoefield R. Sinha S. et al.Serum phosphate and mortality in patients with chronic kidney disease.Clin J Am Soc Nephrol. 2010; 5: 2251-2257Crossref PubMed Scopus (177) Google Scholar, 9Menon V. Greene T. Pereira A.A. et al.Relationship of phosphorus and calcium-phosphorus product with mortality in CKD.Am J Kidney Dis. 2005; 46: 455-463Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar Observational studies assessing the relationship between dietary intervention and serum Pi levels are confounded by the lack of standardization regarding the time of day that serum Pi was assessed.10Selamet U. Tighiouart H. Sarnak M.J. et al.Relationship of dietary phosphate intake with risk of end-stage renal disease and mortality in chronic kidney disease stages 3-5: The Modification of Diet in Renal Disease Study.Kidney Int. 2016; 89: 176-184Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar Serum Pi levels exhibit a well-described daily oscillation in normal and CKD patients.11Becker G.J. Walker R.G. Hewitson T.D. Pedagogos E. Phosphate levels–time for a rethink?.Nephrol Dial Transplant. 2009; 24: 2321-2324Crossref PubMed Scopus (21) Google Scholar Pi peaks between 02:00 and 04:00 (rest phase), and the lowest levels are detected between 08:00 and 10:00 (active phase).11Becker G.J. Walker R.G. Hewitson T.D. Pedagogos E. Phosphate levels–time for a rethink?.Nephrol Dial Transplant. 2009; 24: 2321-2324Crossref PubMed Scopus (21) Google Scholar, 12Jubiz W. Canterbury J.M. Reiss E. Tyler F.H. Circadian rhythm in serum parathyroid hormone concentration in human subjects: correlation with serum calcium, phosphate, albumin, and growth hormone levels.J Clin Invest. 1972; 51: 2040-2046Crossref PubMed Scopus (201) Google Scholar, 13Markowitz M. Rotkin L. Rosen J.F. Circadian rhythms of blood minerals in humans.Science. 1981; 213: 672-674Crossref PubMed Scopus (111) Google Scholar, 14Portale A.A. Halloran B.P. Morris Jr., R.C. Dietary intake of phosphorus modulates the circadian rhythm in serum concentration of phosphorus. Implications for the renal production of 1,25-dihydroxyvitamin D.J Clin Invest. 1987; 80: 1147-1154Google Scholar Most epidemiologic studies have demonstrated that the fasting morning serum Pi concentration is linked to cardiovascular events and mortality. The factors regulating this link, however, are not known.15Chang A.R. Grams M.E. Serum phosphorus and mortality in the Third National Health and Nutrition Examination Survey (NHANES III): effect modification by fasting.Am J Kidney Dis. 2014; 64: 567-573Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 16Tonelli M. Sacks F. Pfeffer M. et al.Relation between serum phosphate level and cardiovascular event rate in people with coronary disease.Circulation. 2005; 112: 2627-2633Crossref PubMed Scopus (705) Google Scholar Plasma Pi concentrations and renal Pi excretion display significant daily oscillations in animals17Bielesz B. Bacic D. Honegger K. et al.Unchanged expression of the sodium-dependent phosphate cotransporter NaPi-IIa despite diurnal changes in renal phosphate excretion.Pflugers Arch. 2006; 452: 683-689Crossref PubMed Scopus (12) Google Scholar, 18Kishikawa T. Takahashi H. Shimazawa E. Ogata E. Diurnal changes in calcium and phosphate metabolism in rats.Horm Metab Res. 1980; 12: 545-551Crossref PubMed Scopus (9) Google Scholar, 19Shinoda H. Seto H. Diurnal rhythms in calcium and phosphate metabolism in rodents and their relations to lighting and feeding schedules.Miner Electrolyte Metab. 1985; 11: 158-166PubMed Google Scholar as well as in humans.12Jubiz W. Canterbury J.M. Reiss E. Tyler F.H. Circadian rhythm in serum parathyroid hormone concentration in human subjects: correlation with serum calcium, phosphate, albumin, and growth hormone levels.J Clin Invest. 1972; 51: 2040-2046Crossref PubMed Scopus (201) Google Scholar, 13Markowitz M. Rotkin L. Rosen J.F. Circadian rhythms of blood minerals in humans.Science. 1981; 213: 672-674Crossref PubMed Scopus (111) Google Scholar, 20Kemp G.J. Blumsohn A. Morris B.W. Circadian changes in plasma phosphate concentration, urinary phosphate excretion, and cellular phosphate shifts.Clin Chem. 1992; 38: 400-402PubMed Google Scholar, 21Ahmad A.M. Hopkins M.T. Fraser W.D. et al.Parathyroid hormone secretory pattern, circulating activity, and effect on bone turnover in adult growth hormone deficiency.Bone. 2003; 32: 170-179Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 22Carruthers B.M. Copp D.H. McIntosh H.W. Diurnal variation in urinary excretion of calcium and phosphate and its relation to blood levels.J Lab Clin Med. 1964; 63: 959-968PubMed Google Scholar, 23Halloran B.P. Portale A.A. Castro M. et al.Serum concentration of 1,25-dihydroxyvitamin D in the human: diurnal variation.J Clin Endocrinol Metab. 1985; 60: 1104-1110Crossref PubMed Scopus (34) Google Scholar The daily oscillation of plasma Pi levels in nocturnal rodents (rats) is roughly inverse to that in humans.14Portale A.A. Halloran B.P. Morris Jr., R.C. Dietary intake of phosphorus modulates the circadian rhythm in serum concentration of phosphorus. Implications for the renal production of 1,25-dihydroxyvitamin D.J Clin Invest. 1987; 80: 1147-1154Google Scholar, 17Bielesz B. Bacic D. Honegger K. et al.Unchanged expression of the sodium-dependent phosphate cotransporter NaPi-IIa despite diurnal changes in renal phosphate excretion.Pflugers Arch. 2006; 452: 683-689Crossref PubMed Scopus (12) Google Scholar, 18Kishikawa T. Takahashi H. Shimazawa E. Ogata E. Diurnal changes in calcium and phosphate metabolism in rats.Horm Metab Res. 1980; 12: 545-551Crossref PubMed Scopus (9) Google Scholar, 19Shinoda H. Seto H. Diurnal rhythms in calcium and phosphate metabolism in rodents and their relations to lighting and feeding schedules.Miner Electrolyte Metab. 1985; 11: 158-166PubMed Google Scholar, 20Kemp G.J. Blumsohn A. Morris B.W. Circadian changes in plasma phosphate concentration, urinary phosphate excretion, and cellular phosphate shifts.Clin Chem. 1992; 38: 400-402PubMed Google Scholar, 24Ix J.H. Anderson C.A. Smits G. et al.Effect of dietary phosphate intake on the circadian rhythm of serum phosphate concentrations in chronic kidney disease: a crossover study.Am J Clin Nutr. 2014; 100: 1392-1397Crossref PubMed Scopus (54) Google Scholar In humans and rodents, plasma Pi levels are decreased during the active phase and increased in the resting phase.14Portale A.A. Halloran B.P. Morris Jr., R.C. Dietary intake of phosphorus modulates the circadian rhythm in serum concentration of phosphorus. Implications for the renal production of 1,25-dihydroxyvitamin D.J Clin Invest. 1987; 80: 1147-1154Google Scholar, 17Bielesz B. Bacic D. Honegger K. et al.Unchanged expression of the sodium-dependent phosphate cotransporter NaPi-IIa despite diurnal changes in renal phosphate excretion.Pflugers Arch. 2006; 452: 683-689Crossref PubMed Scopus (12) Google Scholar, 18Kishikawa T. Takahashi H. Shimazawa E. Ogata E. Diurnal changes in calcium and phosphate metabolism in rats.Horm Metab Res. 1980; 12: 545-551Crossref PubMed Scopus (9) Google Scholar, 19Shinoda H. Seto H. Diurnal rhythms in calcium and phosphate metabolism in rodents and their relations to lighting and feeding schedules.Miner Electrolyte Metab. 1985; 11: 158-166PubMed Google Scholar On the other hand, in humans and rodents, changes in urinary Pi excretion levels are roughly the reverse of the changes in the plasma Pi concentrations.14Portale A.A. Halloran B.P. Morris Jr., R.C. Dietary intake of phosphorus modulates the circadian rhythm in serum concentration of phosphorus. Implications for the renal production of 1,25-dihydroxyvitamin D.J Clin Invest. 1987; 80: 1147-1154Google Scholar, 17Bielesz B. Bacic D. Honegger K. et al.Unchanged expression of the sodium-dependent phosphate cotransporter NaPi-IIa despite diurnal changes in renal phosphate excretion.Pflugers Arch. 2006; 452: 683-689Crossref PubMed Scopus (12) Google Scholar, 18Kishikawa T. Takahashi H. Shimazawa E. Ogata E. Diurnal changes in calcium and phosphate metabolism in rats.Horm Metab Res. 1980; 12: 545-551Crossref PubMed Scopus (9) Google Scholar, 19Shinoda H. Seto H. Diurnal rhythms in calcium and phosphate metabolism in rodents and their relations to lighting and feeding schedules.Miner Electrolyte Metab. 1985; 11: 158-166PubMed Google Scholar, 20Kemp G.J. Blumsohn A. Morris B.W. Circadian changes in plasma phosphate concentration, urinary phosphate excretion, and cellular phosphate shifts.Clin Chem. 1992; 38: 400-402PubMed Google Scholar, 24Ix J.H. Anderson C.A. Smits G. et al.Effect of dietary phosphate intake on the circadian rhythm of serum phosphate concentrations in chronic kidney disease: a crossover study.Am J Clin Nutr. 2014; 100: 1392-1397Crossref PubMed Scopus (54) Google Scholar Prolonged fasting abolishes the nocturnal peak in serum Pi,25Kempson S.A. Shah S.V. Werness P.G. et al.Renal brush border membrane adaptation to phosphorus deprivation: effects of fasting versus low-phosphorus diet.Kidney Int. 1980; 18: 36-47Abstract Full Text PDF PubMed Scopus (38) Google Scholar, 26Min H.K. Jones J.E. Flink E.B. Circadian variations in renal excretion of magnesium, calcium, phosphorus, sodium, and potassium during frequent feeding and fasting.Fed Proc. 1966; 25: 917-921PubMed Google Scholar indicating that dietary intake contributes to the daily changes in serum Pi. Changes in parathyroid hormone (PTH), growth hormone, 1,25(OH)2D3, and fibroblast growth factor 23 (FGF23) cannot fully explain the daily oscillation of plasma Pi concentrations.12Jubiz W. Canterbury J.M. Reiss E. Tyler F.H. Circadian rhythm in serum parathyroid hormone concentration in human subjects: correlation with serum calcium, phosphate, albumin, and growth hormone levels.J Clin Invest. 1972; 51: 2040-2046Crossref PubMed Scopus (201) Google Scholar, 14Portale A.A. Halloran B.P. Morris Jr., R.C. Dietary intake of phosphorus modulates the circadian rhythm in serum concentration of phosphorus. Implications for the renal production of 1,25-dihydroxyvitamin D.J Clin Invest. 1987; 80: 1147-1154Google Scholar, 27Kawai M. Kinoshita S. Shimba S. et al.Sympathetic activation induces skeletal Fgf23 expression in a circadian rhythm-dependent manner.J Biol Chem. 2014; 289: 1457-1466Crossref PubMed Scopus (56) Google Scholar, 28Smith E.R. Cai M.M. McMahon L.P. Holt S.G. Biological variability of plasma intact and C-terminal FGF23 measurements.J Clin Endocrinol Metab. 2012; 97: 3357-3365Crossref PubMed Scopus (154) Google Scholar, 29Trivedi H. Szabo A. Zhao S. et al.Circadian variation of mineral and bone parameters in end-stage renal disease.J Nephrol. 2015; 28: 351-359Crossref PubMed Scopus (23) Google Scholar Pi homeostasis is predominantly regulated by sodium-dependent Pi transporters of the solute carrier family SLC34, including Npt2a, Npt2b, and Npt2c. Npt2a and Npt2c are responsible for reabsorption of approximately 70% to 80% of the Pi filtered by the kidney.3Miyamoto K. Haito-Sugino S. Kuwahara S. et al.Sodium-dependent phosphate cotransporters: lessons from gene knockout and mutation studies.J Pharm Sci. 2011; 100: 3719-3730Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 30Murer H. Hernando N. Forster I. Biber J. Proximal tubular phosphate reabsorption: molecular mechanisms.Physiol Rev. 2000; 80: 1373-1409Crossref PubMed Scopus (449) Google Scholar Small intestinal Npt2b also has an important functional role.3Miyamoto K. Haito-Sugino S. Kuwahara S. et al.Sodium-dependent phosphate cotransporters: lessons from gene knockout and mutation studies.J Pharm Sci. 2011; 100: 3719-3730Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 31Ohi A. Hanabusa E. Ueda O. et al.Inorganic phosphate homeostasis in sodium-dependent phosphate cotransporter Npt2b(+)/(-) mice.Am J Physiol Renal Physiol. 2011; 301: F1105-F1113Crossref PubMed Scopus (37) Google Scholar, 32Sabbagh Y. O'Brien S.P. Song W. et al.Intestinal npt2b plays a major role in phosphate absorption and homeostasis.J Am Soc Nephrol. 2009; 20: 2348-2358Crossref PubMed Scopus (259) Google Scholar, 33Hernando N. Myakala K. Simona F. et al.Intestinal Depletion of NaPi-IIb/Slc34a2 in Mice: Renal and Hormonal Adaptation.J Bone Miner Res. 2015; 30: 1925-1937Crossref PubMed Scopus (45) Google Scholar Serum Pi is a function of Pi homeostasis as well as the balanced movement of Pi between intracellular and extracellular spaces.34Lederer E. Regulation of serum phosphate.J Physiol. 2014; 592: 3985-3995Crossref PubMed Scopus (94) Google Scholar Detailed mechanisms of the cellular Pi shift are unknown, but cellular energy metabolism (ATP and nicotinamide adenine dinucleotide [NAD]+) may be involved in Pi utilization.35Dousa T.P. Modulation of renal Na-Pi cotransport by hormones acting via genomic mechanism and by metabolic factors.Kidney Int. 1996; 49: 997-1004Abstract Full Text PDF PubMed Scopus (24) Google Scholar The role of the SLC34 family in the daily oscillation remains unknown. In a previous study, we investigated a partial hepatectomy-induced hypophosphatemia model and found that the nicotinamide phosphoribosyltransferase (Nampt)/NAD+ system is important for systematic regulation of Npt2a, Npt2b, and Npt2c transporters.36Nomura K. Tatsumi S. Miyagawa A. et al.Hepatectomy-related hypophosphatemia: a novel phosphaturic factor in the liver-kidney axis.J Am Soc Nephrol. 2014; 25: 761-772Crossref PubMed Scopus (35) Google Scholar Nampt acts via enzymatic activity to synthesize nicotinamide mononucleotide and to maintain homeostasis of NAD, which plays a dual role in energy metabolism and biologic signaling.37Revollo J.R. Grimm A.A. Imai S. The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells.J Biol Chem. 2004; 279: 50754-50763Crossref PubMed Scopus (772) Google Scholar, 38Imai S. The NAD World: a new systemic regulatory network for metabolism and aging–Sirt1, systemic NAD biosynthesis, and their importance.Cell Biochem Biophys. 2009; 53: 65-74Crossref PubMed Scopus (159) Google Scholar We hypothesized that the Nampt/NAD+ system controls the daily oscillation. Here we investigated the roles of Npt2 and Nampt in the daily oscillation of plasma Pi concentrations. First, we investigated the daily oscillation of plasma Pi and urinary Pi excretion in wild-type (WT) mice. Plasma Pi levels were lower at 08:00 AM (Zeitgeber time [ZT], light/dark cycle ZT0, lights on; ZT12, lights off) and gradually increased, peaking at around ZT10 (Figure 1a). Thereafter, the plasma Pi concentrations gradually decreased from ZT10 to ZT18. Renal Pi excretion values were highest from ZT10 to ZT14 (Figure 1b). Fractional excretion of phosphate (FEPi, %) values was highest at ZT14 (Figure 1c). We used brush border membrane vesicle (BBMV) total protein as a loading control because actin appears to undergo some time-of-day variation (data not shown). Renal and intestinal BBMV (20 μg/lane) were analyzed by immunoblotting. SDS–PAGE analysis revealed almost the same protein levels in each lane. Npt2a protein levels in the BBMVs gradually decreased from ZT2 to ZT14 and then increased to ZT22 (Figure 1d). The pattern of Npt2c protein levels was not as prominent as that of Npt2a. Daily oscillations of Npt2b protein were similar to those of renal Npt2a (Figure 1e). Renal and intestinal Na/Pi transport activities in the BBMVs were significantly reduced at ZT14 compared with ZT2 (Figure 1f). Plasma PTH and FGF23 levels did not change between ZT2 (rodent rest phase) and ZT14 (rodent active phase; Figure 1g and h). These findings revealed that renal Npt2a protein and intestinal Npt2b protein levels exhibit daily oscillations, like plasma and urinary Pi levels, independent of the plasma FGF23 and PTH levels. Next, we investigated the effect of food deprivation on Pi excretion and plasma Pi levels (Supplementary Figure S1). Animals were analyzed during food deprivation and compared with those fed ad libitum (Supplementary Figure S1A). We analyzed 2 groups (feeding group and food-deprived group) beginning at ZT14 (Supplementary Figure S1B). In the food-deprived group, urinary Pi excretion levels gradually increased. Plasma Pi levels were significantly higher compared with the feeding group in all periods (Supplementary Figure S1B). The Npt2a protein levels were markedly decreased in the food-deprived mice (Supplementary Figure S1C). These findings suggest that the daily oscillation of plasma Pi concentrations depends on food intake, which is consistent with previous findings.39Trohler U. Bonjour J.P. Fleisch H. Plasma level and renal handling of Pi: effect of overnight fasting with and without Pi supply.Am J Physiol. 1981; 241: F509-F516PubMed Google Scholar To investigate whether renal Npt2 proteins are involved in the daily oscillation of the plasma Pi concentration, we analyzed the daily oscillations of Npt2a−/−, Npt2a−/−/Npt2c−/−, and intestine-specific Npt2b deletion mice (Npt2bflox/flox-vCre) (Figure 2). Food intake behavior did not differ between groups (Figure 2c). Npt2a−/− mice have hypophosphatemia and hyperphosphaturia. During the diurnal phase (ZT2−ZT10), the increase in the plasma Pi concentration observed in Npt2a+/+mice was not observed in Npt2a−/− mice (Figure 2a). During the active phase (ZT14−ZT22), however, the reduced plasma Pi concentration in Npt2a+/+ mice was also observed in Npt2a−/− mice (Figure 2a). In contrast, during the active phase, renal Pi excretion levels were significantly increased in Npt2a−/− mice and Npt2a+/+ mice (Figure 2b). Npt2a protein levels in Npt2a+/+ mice were markedly decreased at ZT14 compared with ZT2 (Figure 2d). Npt2c protein levels were highest at ZT2 and ZT14 in Npt2a−/− mice. Unlike Npt2a+/+ mice, Npt2a−/− mice showed no increase in the plasma Pi concentration during the rest phase (ZT2−ZT10), whereas the plasma Pi concentration was reduced during the active phase. We further investigated the role of intestinal Npt2b in the daily oscillation of plasma Pi concentrations using Npt2bflox/flox-vCre mice (intestine-specific Npt2b deletion mice). Npt2bflox/flox-vCre mice had normal plasma Pi levels, but decreased renal Pi excretion, as reported previously.33Hernando N. Myakala K. Simona F. et al.Intestinal Depletion of NaPi-IIb/Slc34a2 in Mice: Renal and Hormonal Adaptation.J Bone Miner Res. 2015; 30: 1925-1937Crossref PubMed Scopus (45) Google Scholar Our established Npt2bflox/flox-vCre mice, however, had lower plasma Pi levels than vCre+ (control) mice at only 8 weeks. Increased plasma Pi concentrations were observed in intestinal vCre+ mice and intestinal Npt2bflox/flox-vCre mice during ZT2 to ZT10 (Figure 2e). Furthermore, plasma Pi concentrations were reduced during ZT14 to ZT22 in vCre+ mice and Npt2bflox/flox-vCre mice (Figure 2e). Urinary Pi excretion was suppressed in Npt2bflox/flox-vCre mice compared with vCre+ mice (Figure 2f). The daily oscillation pattern of plasma and urine Pi in Npt2bflox/flox-vCre mice was similar to that in intestinal Npt2b vCre+ mice. Next, we analyzed Npt2a−/−/Npt2c−/− mice. The plasma Pi concentration was markedly decreased and the renal Pi excretion was conversely increased (Figure 2g and h), as reported previously.40Segawa H. Onitsuka A. Furutani J. et al.Npt2a and Npt2c in mice play distinct and synergistic roles in inorganic phosphate metabolism and skeletal development.Am J Physiol Renal Physiol. 2009; 297: F671-F678Crossref PubMed Scopus (115) Google Scholar The daily oscillation of the plasma Pi concentration observed in WT mice (Figure 1a) was not observed in Npt2a−/−/Npt2c−/− mice. These findings suggest that the 2 transporters are indispensable to the daily oscillation of plasma Pi concentration. We then investigated the factors controlling the daily oscillation of the plasma Pi concentration. In the small intestine and kidney, NAD levels were significantly increased at ZT14 compared with ZT2 (Figure 3a and b). NAD levels in the liver, on the other hand, were significantly increased at ZT2 compared with ZT14. These data suggest that cellular NAD levels affect Npt2a and Npt2b levels (Figure 3c). We further investigated the effect of phosphaturic factors (Pi load, PTH, and nicotinamide [NAM]) on Pi excretion between the rest and active phases (Figure 3d). Pi load significantly stimulated renal Pi excretion to the same extent in the rest and active phases (Figure 3e). PTH injection significantly increased the Pi load in the rest phase, but not the active phase (Figure 3f). Changes in the cellular NAD concentration affect PTH responsiveness.41Berndt T.J. Knox F.G. Kempson S.A. Dousa T.P. Nicotinamide adenine dinucleotide and renal response to parathyroid hormone.Endocrinology. 1981; 108: 2005-2007Crossref PubMed Scopus (17) Google Scholar Next, we analyzed the effect of NAM on Pi excretion in the rest and active phases (Figure 3g and h). Injection of NAM increased the cellular NAD concentration (Figure 3g). In the active phase, NAM injection did not affect phosphaturic activity (Figure 3h). Npt2a protein levels were decreased in the rest phase, but not in the active phase (data not shown). These findings indicate that the effect of NAM differs between the rest and active phases. The Nampt enzyme is a rate-limiting step of cellular NAD synthesis and has a circadian rhythm.37Revollo J.R. Grimm A.A. Imai S. The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells.J Biol Chem. 2004; 279: 50754-50763Crossref PubMed Scopus (772) Google Scholar, 38Imai S. The NAD World: a new systemic regulatory network for metabolism and aging–Sirt1, systemic NAD biosynthesis, and their importance.Cell Biochem Biophys. 2009; 53: 65-74Crossref PubMed Scopus (159) Google Scholar We analyzed the effect of a Nampt inhibitor (FK866) on C57BL6 mice (WT) (Figure 4). We injected FK866 into WT mice at ZT0 (rest phase) and ZT16 (active phase), and measured cellular NAD concentrations 4 hours later (Figure 4a). FK866 treatment significantly decreased renal NAD concentrations at ZT20 (active phase), but not at ZT4 (rest phase) (Figure 4b). Intestinal NAD contents tended to decrease similarly (Figure 4c). At ZT4, the liver NAD contents were significantly decreased (Figure 4d). Thus, fluctuations in the NAD levels in the intestine and kidney tended to be opposite those in the liver. FK866 treatment during the rest phase did not affect renal Pi excretion or plasma Pi levels. In contrast, FK866 significantly blocked Pi excretion at ZT16 to ZT20 (active phase) (Figure 4e). In addition, FK866 significantly increased plasma Pi concentrations at ZT20. These observations suggest that Nampt inhibition is involved in the daily oscillation of plasma Pi and Pi excretion. Inhibition of the nocturnal (active phase) increase in Nampt activity prevented the reduction of renal Npt2a and intestinal Npt2b protein levels during the active phase (Figure 4f). The results shown in Figures 3 and 4 suggest that renal and intestinal Pi transport is controlled by the Nampt activity in the resting and active phases, respectively. In a previous study, we found that Pi release from the liver may contribute to maintaining the plasma Pi concentration.36Nomura K. Tatsumi S. Miyagawa A. et al.Hepatectomy-related hypophosphatemia: a novel phosphaturic factor in the liver-kidney axis.J Am Soc Nephrol. 2014; 25: 761-772Crossref PubMed Scopus (35) Google Scholar Liver NAD levels exhibited a prominent daily rhythm that depended on food intake, as reported previously.42Woller A. Duez H. Staels B. Lefranc M. A Mathematical Model of the Liver Circadian Clock Linking Feeding and Fasting Cycles to Clock Function.Cell Rep. 2016; 17: 1087-1097Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Next, we investigated liver-specific Nampt-deletion mice (Namptflox/flox-aCre). Liver Nampt levels exhibit a remarkable diurnal rhythm.43Ramsey K.M. Yoshino J. Brace C.S. et al.Circadian clock feedback cycle through NAMPT-mediated NAD+ biosynthesis.Science. 2009; 324: 651-654Crossref PubMed Scopus (849) Google Scholar The body weight of liver-specific Namptflox/flox-aCre mice was decreased compared with Namptflox/flox mice (Figure 5a ). In liver-specific Namptflox/flox-aCre mice, the liver NAD levels were significantly decreased during the rest phase (ZT6), bu
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