Effect of CKD-MBD therapies on the gastrointestinal expression of phosphate transporters

Background: Chronic kidney disease-mineral and bone disorder (CKD-MBD) is prevalent and encompasses biochemical abnormalities, bone alterations, and vascular calcifications. CKD-MBD treatments include phosphate binders and calcimimetics that effectively lower phosphorus and PTH, respectively, but the impact of these treatments on phosphate transporters in the gastrointestinal tract is still unknown. NaPi2B is considered the primary intestinal phosphate transporter. However, NaPi2B inhibition shows limited effectiveness, thus the importance of PIT1 and PIT2 requires further investigation. Methods: We tested the effects phosphate binders (ferric citrate (FC) and calcium gluconate (Ca)) and the calcimimetic KP2326 (KP) in (Cy/+ male rat; CKD) and untreated normal littermates (NL). Treatments lasted 10 weeks until euthanasia at 28 weeks (moderate-to-advanced CKD), where we collected mucosa samples from duodenum, jejunum, and ileum. Blood was collected for biochemistry measurements. Total RNA was isolated, and qPCR performed to assess phosphate transporters expression (NaPi2B, PIT1, PIT2) normalized to b-actin. Results were analyzed via 2-way ANOVA. Results: As expected, CKD had abnormal plasma concentrations of phosphorus, PTH, and FGF23. FC and KP effectively lowered phosphorus. KP and Ca lowered PTH, but Ca increased FGF23. NaPi2B was expressed in the duodenum and jejunum but not in the ileum, and its expression was upregulated with FC compared to NL. PIT1 was expressed in all segments, but the expression was the highest in the ileum, while PIT2 was constitutively expressed in all segments. Ca led to higher PIT1 and PIT2 expression in the duodenum and jejunum compared to NL, CKD, or KP. KP led to higher expression of PIT1 in the ileum compared to CKD, FC, and Ca. Conclusions: CKD-MBD therapies differentially impacted biochemistries and phosphate transporters expression. The effect of Ca on gastrointestinal expression of PIT1 and PIT2 may explain the higher plasma phosphorus and should be further explored.

Integrative transcriptomic and metabolomic analysis of D-leaf of seven pineapple varieties differing in N-P-K% contents

Background Pineapple (Ananas comosus L. Merr.) is the third most important tropical fruit in China. In other crops, farmers can easily judge the nutritional requirements from leaf color. However, concerning pineapple, it is difficult due to the variation in leaf color of the cultivated pineapple varieties. A detailed understanding of the mechanisms of nutrient transport, accumulation, and assimilation was targeted in this study. We explored the D-leaf nitrogen (N), phosphorus (P), and potassium (K) contents, transcriptome, and metabolome of seven pineapple varieties. Results Significantly higher N, P, and K% contents were observed in Bali, Caine, and Golden pineapple. The transcriptome sequencing of 21 libraries resulted in the identification of 14,310 differentially expressed genes in the D-leaves of seven pineapple varieties. Genes associated with N transport and assimilation in D-leaves of pineapple was possibly regulated by nitrate and ammonium transporters, and glutamate dehydrogenases play roles in N assimilation in arginine biosynthesis pathways. Photosynthesis and photosynthesis-antenna proteins pathways were also significantly regulated between the studied genotypes. Phosphate transporters and mitochondrial phosphate transporters were differentially regulated regarding inorganic P transport. WRKY, MYB, and bHLH transcription factors were possibly regulating the phosphate transporters. The observed varying contents of K% in the D-leaves was associated to the regulation of K+ transporters and channels under the influence of Ca2+ signaling. The UPLC-MS/MS analysis detected 873 metabolites which were mainly classified as flavonoids, lipids, and phenolic acids. Conclusions These findings provide a detailed insight into the N, P, K% contents in pineapple D-leaf and their transcriptomic and metabolomic signatures.

Analysis of sex difference in the tubular reabsorption of lithium in rats

Lithium is used in the treatment of bipolar disorder. We previously demonstrated that two types of transporters mediate the tubular reabsorption of lithium in rats, and suggested that sodium-dependent phosphate transporters play a role in lithium reabsorption with high affinity. In the present study, we examined sex differences in lithium reabsorption in rats. When lithium chloride was infused at 60 µg/min, creatinine clearance and the renal clearance of lithium were lower, and the plasma concentration of lithium was higher in female rats. These values reflected the higher fractional reabsorption of lithium in female rats. In rats infused with lithium chloride at 6 µg/min, the pharmacokinetic parameters of lithium examined were all similar in both sexes. The fractional reabsorption of lithium was decreased by foscarnet, a representative inhibitor of sodium-dependent phosphate transporters, in male and female rats when lithium chloride was infused at the low rate. Among the candidate transporters mediating lithium reabsorption examined herein, the mRNA expression of only PiT2, a sodium-dependent phosphate transporter, exhibited sexual dimorphism. The present results demonstrated sex differences in the tubular reabsorption of lithium with low affinity in rats.

Key computational findings reveal proton transfer as driving the functional cycle in the phosphate transporter PiPT

Phosphate is an indispensable metabolite in a wide variety of cells and is involved in nucleotide and lipid synthesis, signaling, and chemical energy storage. Proton-coupled phosphate transporters within the major facilitator family are crucial for phosphate uptake in plants and fungi. Similar proton-coupled phosphate transporters have been found in different protozoan parasites that cause human diseases, in breast cancer cells with elevated phosphate demand, in osteoclast-like cells during bone reabsorption, and in human intestinal Caco2BBE cells for phosphate homeostasis. However, the mechanism of proton-driven phosphate transport remains unclear. Here, we demonstrate in a eukaryotic, high-affinity phosphate transporter from Piriformospora indica (PiPT) that deprotonation of aspartate 324 (D324) triggers phosphate release. Quantum mechanics/molecular mechanics molecular dynamics simulations combined with free energy sampling have been employed here to identify the proton transport pathways from D324 upon the transition from the occluded structure to the inward open structure and phosphate release. The computational insights so gained are then corroborated by studies of D45N and D45E amino acid substitutions via mutagenesis experiments. Our findings confirm the function of the structurally predicted cytosolic proton exit tunnel and suggest insights into the role of the titratable phosphate substrate.

FC 004PHOSPHATE FACILITATES PHOSPHATURIA BY PIT-2-MEDIATED ACTIVATION OF ERK1/2 SIGNALLING INTERNALIZING NAPI-2A FROM THE APICAL BRUSH BORDER MEMBRANE INDEPENDENT OF FGF23

Background and Aims Increased phosphate load stimulates the secretion of fibroblast growth factor (FGF) 23 in the bone leading to decreased phosphate reabsorption in the kidney. FGF23 activates FGFR1/Klotho/ERK1/2 signalling in proximal tubule cells to suppress type II sodium phosphate transporters NaPi-2a and NaPi-2c in the apical brush border membrane (BBM) resulting in lower serum phosphate levels. The type III sodium-dependent phosphate transporters PiT-1 and PiT-2 are expressed in key organs of phosphate regulation and were shown to activate ERK1/2 in osseous cells in the presence of high extracellular phosphate. Furthermore, PiT-2 was shown to be responsible for the phosphate-dependent FGF23 secretion in bone cells. Whether phosphate itself can be sensed by kidney cells and stimulate its own excretion remains unknown. The aim of our study was to examine the molecular mechanism regulating renal phosphate transport in the setting of chronic oral phosphate loading in mice and to analyse phosphate sensing as well as phosphaturic actions of phosphate itself independent of FGF23. Method First, eight-week-old male C57BL/6 wildtype mice were fed a 2% high phosphate diet (HPD) or a 0.8% normal phosphate diet (NPD). Mice were sacrificed after six months and blood and urine were collected to determine parameters of phosphate homeostasis. Kidneys were isolated to evaluate the HPD-induced regulation of phosphate transporters by qPCR, immunoblot and histological analyses. Second, murine proximal tubule (mPT) cells were stimulated with either phosphate or FGF23 in the presence or absence of Foscarnet, as an inhibitor of phosphate transporters, to verify the molecular mechanism of phosphate sensing. Results Although, HPD caused significantly elevated circulating levels of intact FGF23 which resulted in hyperphosphaturia, serum phosphate levels were still enhanced compared to NPD-fed mice. Renal Klotho protein expression was significantly reduced in HPD mice and histological staining demonstrated lower Klotho accumulation in proximal and distal tubule cells, while FGFR1 was not altered. The FGF23/Klotho/FGFR1 downstream pathway revealed neither a clear activation of the ERK1/2 signalling pathway nor induction of the transcription factor Egr-1 due to HPD. Nevertheless, NaPi-2a mRNA expression was significantly reduced in HPD-fed mice compared to NPD group and NaPi-2c was unchanged. The amount of NaPi-2a protein in isolated BBM vesicles of HPD-fed mice was lower compared to NPD and immunofluorescent staining confirmed the internalisation of NaPi-2a from the apical BBM. Among the type III sodium-dependent phosphate cotransporters, renal PiT-1 mRNA expression was not altered in HPD-fed mice, but PiT-2 was significantly increased compared to NPD group and immunofluorescent staining revealed an enhanced localization of PiT-2 on the basolateral membrane of proximal tubule cells. Stimulation of mPTs with phosphate or FGF23 increased the expression of PiT-2, induced the phosphorylation of ERK1/2 and decreased NaPi-2a in vitro. The pre-treatment with Foscarnet blunted the phosphate-mediated activation of ERK1/2 signalling pathway, but not the FGF23-induced effects, suggesting a direct phosphate transporter-regulating mechanism of high phosphate in renal proximal tubule cells. Conclusion A chronic high dietary intake of phosphate results in downregulation of renal Klotho causing hyperphosphatemia, suggesting in part a renal resistance of FGF23/Klotho signalling pathway. However, HPD-induced internalization NaPi-2a from the apical BBM pointing to an FGF23-independent mechanism regulating phosphate reabsorption. Our data indicate that in the settings of high phosphate-mediated renal resistance of FGF23, phosphate itself may stimulate its urinary secretion via PiT-2-mediated activation of ERK1/2 signalling pathway which results in NaPi-2a downregulation and hyperphosphaturia independent of FGF23.

Novel Mineral Regulatory Pathways in Ovine Pregnancy: I. Phosphate, Klotho Signaling, and Sodium Dependent Phosphate Transporters

Appropriate mineralization of the fetal skeleton requires an excess of phosphate in the fetus compared to the mother. However, mechanisms for placental phosphate transport are poorly understood. This study aimed to identify phosphate regulatory pathways in ovine endometria and placentae throughout gestation. Suffolk ewes were bred with fertile rams upon visual detection of estrus (Day 0). On Days 9, 12, 17, 30, 70, 90, 110, and 125 of pregnancy (n = 3–14/Day), ewes were euthanized and hysterectomized. Phosphate abundance varied across gestational days in uterine flushings, allantoic fluid, and homogenized endometria and placentae (P < 0.05). The expression of mRNAs for sodium-dependent phosphate transporters (SLC20A1 and SLC20A2) and klotho signaling mediators (FGF7, FGF21, FGF23, FGFR1–4, KL, KLB, ADAM10, and ADAM17) were quantified by qPCR. Day 17 conceptus tissue expressed SLC20A1, SLC20A2, KLB, FGF7, FGF21, FGF23, FGFR1, and FGFR2 mRNAs. Both sodium-dependent phosphate transporters and klotho signaling mediators were expressed in endometria and placentae throughout gestation. Gestational day influenced the expression of SLC20A1, ADAM10, ADAM17, FGF21, FGFR1, and FGFR3 mRNAs in both endometria and placentae (P < 0.05). Gestational day influenced endometrial expression of FGF7 (P < 0.001), and placental expression of FGF23 (P < 0.05). Immunohistochemistry confirmed that both FGF23 and KL proteins were expressed in endometria and placentae throughout gestation. The observed spatiotemporal profile of KL-FGF signaling suggests a potential role in the establishment of pregnancy and regulation of fetal growth. This study provides a platform for further mechanistic investigation into the role for KL-FGF signaling in the regulation of phosphate transport at the ovine maternal-conceptus interface.

Phosphate Uptake and Transport in Plants: An Elaborate Regulatory System

Phosphorus (P) is an essential macronutrient for plant growth and development. Low phosphate (Pi) availability is a limiting factor for plant growth and yield. To cope with a complex and changing environment, plants have evolved elaborate mechanisms for regulating Pi uptake and use. Recently, the molecular mechanisms of plant Pi signaling have become clearer. Plants absorb Pi from the soil through their roots and transfer Pi to various organs or tissues through phosphate transporters, which are precisely controlled at the transcript and protein levels. Here, we summarize the recent progress on the molecular regulatory mechanism of phosphate transporters in Arabidopsis and rice, including the characterization of functional transporters, regulation of transcript levels, protein localization, and turnover of phosphate transporters. A more in-depth understanding of plant adaptation to a changing Pi environment will facilitate the genetic improvement of plant P efficiency.