Probiotic treatment with Bifidobacterium animalis subsp. lactis LKM512 + arginine improves cognitive flexibility in middle-aged mice.

Cognitive flexibility, the ability of adapting to an ever-changing environment, declines with aging and impaired in early stages of dementia. Although recent studies have indicated there is a relationship between the intestinal microbiota and cognitive function, few studies have shown relationships between intestinal microbiota and cognitive flexibility because of limited behavioural tasks in mice. We recently established a novel cognitive flexibility task for mice using a touchscreen operant apparatus and found that probiotic treatment with a mixture of Bifidobacterium animalis subsp. lactis LKM512 and arginine improved cognitive flexibility in young adult mice. To confirm the effects of the probiotic treatment on cognitive flexibility and to determine whether it is effective even in older age, we here examined the effects of long-term treatment with Bifidobacterium animalis subsp. lactis LKM512 and arginine on cognitive flexibility in middle-aged mice. From 8 to 15 months of age, mice received LKM + Arg or vehicle (controls) orally three times per week and were subjected to the cognitive flexibility task at 13-15 months old. In one of indices of cognitive flexibility, both Bifidobacterium animalis subsp. lactis LKM512 and arginine-treated mice and vehicle-treated mice showed progressively improved performance by repeating reversal tasks, with a small trend that Bifidobacterium animalis subsp. lactis LKM512 and arginine-treated mice showed better learning performance through reversal phases. With respect to the other index of cognitive flexibility, Bifidobacterium animalis subsp. lactis LKM512 and arginine-treated mice showed significantly fewer error choices than control mice at the reversal phase, i.e. Bifidobacterium animalis subsp. lactis LKM512 and arginine improved the performance of behavioural sequencing acquired in the previous phase, which allowed Bifidobacterium animalis subsp. lactis LKM512 and arginine-treated mice to show an early onset of shift to reversal contingency. Taken together, long-term treatment with Bifidobacterium animalis subsp. lactis LKM512 and arginine was found to improve cognitive flexibility in middle-aged mice, indicating that probiotic treatment might contribute to prevention of age-related cognitive decline.

Bifidobacterium animalis subsp. lactis and arginine mixture intake improves cognitive flexibility in mice.

The relationship between intestinal microbiota and cognitive function has been investigated as one of the major topics within the intestinal microbiota-gut-brain axis. Although an increasing number of studies have demonstrated an improvement in learning and memory when using probiotics or prebiotics, to date, there are no studies that target the cognitive flexibility observed in the early stages of several neuropsychiatric diseases, including dementia. We have recently developed a novel behavioral task using the touchscreen operant system to assess cognitive flexibility. We found that the disruption of the intestinal microbiota in mice induced a decline in cognitive flexibility. In the present study, we investigated the effects of treatments consisting of Bifidobacterium animalis subsp. lactis and arginine (Bifal + Arg), which promote the production of intestinal bacterial polyamine, on cognitive flexibility in the mouse model. Male C57BL6 mice orally treated with Bifal + Arg three times a week gradually decreased the 1st-choice incorrect diagonal rate with repeated reversals compared with the control group. Furthermore, in serial reversal phases, Bifal + Arg-treated mice shifted to the behavior of choosing a new correct spot more quickly after the reversal, and this was faster with repeated reversals. These results indicate that this treatment adapts to change and improves cognitive flexibility. This is the first report to show that intestinal environmental control, including probiotics and prebiotics, improves cognitive flexibility in mice.

Impact of Intestinal Microbiota on Cognitive Flexibility by a Novel Touch Screen Operant System Task in Mice.

Cognitive flexibility is the ability to rapidly adapt to a constantly changing environment. It is impaired by aging as well as in various neurological diseases, including dementia and mild cognitive impairment. In rodents, although many behavioral test protocols have been reported to assess learning and memory dysfunction, few protocols address cognitive flexibility. In this study, we developed a novel cognitive flexibility test protocol using touch screen operant system. This test comprises a behavioral sequencing task, in which mice are required to discriminate between the "rewarded" and "never-rewarded" spots and shuttle between the two distantly positioned rewarded spots, and serial reversals, in which the diagonal spatial patterns of rewarded and never-rewarded spots were reversely changed repetitively. Using this test protocol, we demonstrated that dysbiosis treated using streptomycin induces a decline in cognitive flexibility, including perseveration and persistence. The relative abundances of Firmicutes and Bacteroides were lower and higher, respectively, in the streptomycin-treated mice with less cognitive flexibility than in the control mice. This is the first report to directly show that intestinal microbiota affects cognitive flexibility.

Role of sodium-dependent Pi transporter/Npt2c on Pi homeostasis in klotho knockout mice different properties between juvenile and adult stages.

SLC34A3/NPT2c/NaPi-2c/Npt2c is a growth-related NaPi cotransporter that mediates the uptake of renal sodium-dependent phosphate (Pi). Mutation of human NPT2c causes hereditary hypophosphatemic rickets with hypercalciuria. Mice with Npt2c knockout, however, exhibit normal Pi metabolism. To investigate the role of Npt2c in Pi homeostasis, we generated α-klotho-/- /Npt2c-/- (KL2cDKO) mice and analyzed Pi homeostasis. α-Klotho-/- (KLKO) mice exhibit hyperphosphatemia and markedly increased kidney Npt2c protein levels. Genetic disruption of Npt2c extended the lifespan of KLKO mice similar to that of α-Klotho-/- /Npt2a-/- mice. Adult KL2cDKO mice had hyperphosphatemia, but analysis of Pi metabolism revealed significantly decreased intestinal and renal Pi (re)absorption compared with KLKO mice. The 1,25-dihydroxy vitamin D3 concentration was not reduced in KL2cDKO mice compared with that in KLKO mice. The KL2cDKO mice had less severe soft tissue and vascular calcification compared with KLKO mice. Juvenile KL2cDKO mice had significantly reduced plasma Pi levels, but Pi metabolism was not changed. In Npt2cKO mice, plasma Pi levels began to decrease around the age of 15 days and significant hypophosphatemia developed within 21 days. The findings of the present study suggest that Npt2c contributes to regulating plasma Pi levels in the juvenile stage and affects Pi retention in the soft and vascular tissues in KLKO mice.

Systemic network for dietary inorganic phosphate adaptation among three organs.

Inorganic phosphate (Pi) secretion from the salivary glands and dietary Pi are key Pi sources. The regulatory mechanisms of Pi homeostasis in the salivary glands are unknown. We investigated how salivary Pi concentrations are regulated by dietary Pi in mouse models. Dietary manipulation significantly changed the levels of Npt2b protein in the salivary gland ductal cells. In addition, rapid feeding on a high-Pi diet increased the saliva Pi concentrations and led to rapid endocytosis of Npt2b in the apical membranes of the duct cells. Global Npt2b± mice exhibited increased salivary Pi concentrations and intestine-specific deletion of Npt2b after high Pi loading increased the salivary Pi concentrations. These findings indicate that Npt2b levels in the salivary glands affect the salivary Pi concentration and are regulated by dietary Pi. Intestinal Npt2b levels might also affect salivary Pi concentrations as well as renal Pi excretion. These findings suggest Pi is endogenously recycled by salivary Pi secretion, intestinal Pi absorption, and renal Pi excretion.

A Role of Intestinal Alkaline Phosphatase 3 (Akp3) in Inorganic Phosphate Homeostasis.

BACKGROUND/AIMS: Hyperphosphatemia is a serious complication of late-stage chronic kidney disease (CKD). Intestinal inorganic phosphate (Pi) handling plays an important role in Pi homeostasis in CKD. We investigated whether intestinal alkaline phosphatase 3 (Akp3), the enzyme that hydrolyzes dietary Pi compounds, is a target for the treatment of hyperphosphatemia in CKD. METHODS: We investigated Pi homeostasis in Akp3 knockout mice (Akp3-/-). We also studied the progression of renal failure in an Akp3-/- mouse adenine treated renal failure model. Plasma, fecal, and urinary Pi and Ca concentration were measured with commercially available kit, and plasma fibroblast growth factor 23, parathyroid hormone, and 1,25(OH)2D3 concentration were measured with ELISA. Brush border membrane vesicles were prepared from mouse intestine using the Ca2+ precipitation method and used for Pi transport activity and alkaline phosphatase activity. In vivo intestinal Pi absorption was measured with oral 32P administration. RESULTS: Akp3-/- mice exhibited reduced intestinal type II sodium-dependent Pi transporter (Npt2b) protein levels and Na-dependent Pi co-transport activity. In addition, plasma active vitamin D levels were significantly increased in Akp3-/- mice compared with wild-type animals. In the adenine-induced renal failure model, Akp3 gene deletion suppressed hyperphosphatemia. CONCLUSION: The present findings indicate that intestinal Akp3 deletion affects Na+-dependent Pi transport in the small intestine. In the adenine-induced renal failure model, Akp3 is predicted to be a factor contributing to suppression of the plasma Pi concentration.

Eldecalcitol Causes FGF23 Resistance for Pi Reabsorption and Improves Rachitic Bone Phenotypes in the Male Hyp Mouse.

X-linked hypophosphatemia (XLH), the most common form of inheritable rickets, is caused by inactivation of phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and leads to fibroblast growth factor (FGF) 23-dependent renal inorganic phosphate (Pi) wasting. In the present study, we investigated whether maintaining Pi homeostasis with a potent vitamin D3 analog, eldecalcitol [1α,25-dihydroxy-2ß-(3-hydroxypropyloxy) vitamin D3; ED71], could improve hypophosphatemic rickets in a murine model of XLH, the Hyp mouse. Vehicle, ED71, or 1,25-dihydroxyvitamin D was subcutaneously injected five times weekly in wild-type (WT) and Hyp mice for 4 weeks, from 4 to 8 weeks of age. Injection of ED71 into WT mice suppressed the synthesis of renal 1,25-dihydroxyvitamin D and promoted phosphaturic activity. In contrast, administration of ED71 to Hyp mice completely restored renal Pi transport and NaPi-2a protein levels, although the plasma-intact FGF23 levels were further increased. In addition, ED71 markedly increased the levels of the scaffold proteins, renal sodium-hydrogen exchanger regulatory factor 1, and ezrin in the Hyp mouse kidney. Treatment with ED71 increased the body weight and improved hypophosphatemia, the bone volume/total volume, bone mineral content, and growth plate structure in Hyp mice. Thus, ED71 causes FGF23 resistance for phosphate reabsorption and improves rachitic bone phenotypes in Hyp mice. In conclusion, ED71 has opposite effects on phosphate homeostasis in WT and Hyp mice. Analysis of Hyp mice treated with ED71 could result in an additional model for elucidating PHEX abnormalities.

The sodium phosphate cotransporter family and nicotinamide phosphoribosyltransferase contribute to the daily oscillation of plasma inorganic phosphate concentration.

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.

Effect of Npt2b deletion on intestinal and renal inorganic phosphate (Pi) handling.

BACKGROUND: Hyperphosphatemia is common in chronic kidney disease and is associated with morbidity and mortality. The intestinal Na+-dependent phosphate transporter Npt2b is thought to be an important molecular target for the prevention of hyperphosphatemia. The role of Npt2b in the net absorption of inorganic phosphate (Pi), however, is controversial. METHODS: In the present study, we made tamoxifen-inducible Npt2b conditional knockout (CKO) mice to analyze systemic Pi metabolism, including intestinal Pi absorption. RESULTS: Although the Na+-dependent Pi transport in brush-border membrane vesicle uptake levels was significantly decreased in the distal intestine of Npt2b CKO mice compared with control mice, plasma Pi and fecal Pi excretion levels were not significantly different. Data obtained using the intestinal loop technique showed that Pi uptake in Npt2b CKO mice was not affected at a Pi concentration of 4 mM, which is considered the typical luminal Pi concentration after meals in mice. Claudin, which may be involved in paracellular pathways, as well as claudin-2, 12, and 15 protein levels were significantly decreased in the Npt2b CKO mice. Thus, Npt2b deficiency did not affect Pi absorption within the range of Pi concentrations that normally occurs after meals. CONCLUSION: These findings indicate that abnormal Pi metabolism may also be involved in tight junction molecules such as Cldns that are affected by Npt2b deficiency.

Effect of Osteocyte-Ablation on Inorganic Phosphate Metabolism: Analysis of Bone-Kidney-Gut Axis.

In response to kidney damage, osteocytes increase the production of several hormones critically involved in mineral metabolism. Recent studies suggest that osteocyte function is altered very early in the course of chronic kidney disease. In the present study, to clarify the role of osteocytes and the canalicular network in mineral homeostasis, we performed four experiments. In Experiment 1, we investigated renal and intestinal Pi handling in osteocyte-less (OCL) model mice [transgenic mice with the dentin matrix protein-1 promoter-driven diphtheria toxin (DT)-receptor that were injected with DT]. In Experiment 2, we administered granulocyte colony-stimulating factor to mice to disrupt the osteocyte canalicular network. In Experiment 3, we investigated the role of osteocytes in dietary Pi signaling. In Experiment 4, we analyzed gene expression level fluctuations in the intestine and liver by comparing mice fed a high Pi diet and OCL mice. Together, the findings of these experiments indicate that osteocyte ablation caused rapid renal Pi excretion (P < 0.01) before the plasma fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH) levels increased. At the same time, we observed a rapid suppression of renal Klotho (P < 0.01), type II sodium phosphate transporters Npt2a (P < 0.01) and Npt2c (P < 0.05), and an increase in intestinal Npt2b (P < 0.01) protein. In OCL mice, Pi excretion in feces was markedly reduced (P < 0.01). Together, these effects of osteocyte ablation are predicted to markedly increase intestinal Pi absorption (P < 0.01), thus suggesting that increased intestinal Pi absorption stimulates renal Pi excretion in OCL mice. In addition, the ablation of osteocytes and feeding of a high Pi diet affected FGF15/bile acid metabolism and controlled Npt2b expression. In conclusion, OCL mice exhibited increased renal Pi excretion due to enhanced intestinal Pi absorption. We discuss the role of FGF23-Klotho on renal and intestinal Pi metabolism in OCL mice.

[New Developments in CKD-MBD. Molecular mechanisms of intestinal and renal phosphate transport].

Intestinal inorganic phosphate (Pi) absorption and renal Pi reabsorption are regulated by parathyroid hormone, fibroblast growth factor 23, or 1,25-dihydroxyvitamin D, which modulate the expression of Pi transporters. Identification, functional characterization and analysis of knockout mice of Pi transporters contributed to clarify the role of each transporter in Pi homeostasis. Furthermore, the molecular mechanisms will lead to the development of new pharmaceutical research approaches.

Effect of dietary components on renal inorganic phosphate (Pi) excretion induced by a Pi-depleted diet.

Dietary inorganic phosphate (Pi) is the most important factor in the regulation of renal Pi excretion. Recent studies suggest the presence of an enteric-renal signaling axis for dietary Pi as well as the existence of a mechanism by which the intestine detects changes in luminal Pi concentrations. The mechanisms of intestinal Pi sensing, however, are unknown. In the present study, we focused on Pi depletion signals and investigated the effects of dietary components on intestinal Pi sensing. After feeding rats experimental diets for 3 days, we investigated urinary Pi excretion and plasma biochemical parameters. Renal Pi excretion was suppressed in rats fed a low-Pi diet (0.02% Pi). Elimination of dietary calcium (Ca) completely blocked the suppression of Pi excretion, suggesting that the presence of Ca is essential for the Pi depletion signal. Furthermore, a minimum Ca content of more than 0.02% was necessary for the Pi depletion signal. Magnesium, lanthanum, and strontium, which are agonists of calcium sensing receptor, instead of Ca, reduced Pi excretion. Therefore, dietary Ca appears to be important for the Pi depletion-sensing mechanism in the gastrointestinal tract. In addition, the calcium sensing receptor may be involved in the Pi depletion signal.