Sodium-dependent Phosphate Cotransporter Research Articles

Hypoxia-Inducible Factor-1α Regulates High Phosphate-Induced Vascular Calcification via Type III Sodium-Dependent Phosphate Cotransporter 1.

Vascular calcification (VC) has a high incidence in patients with chronic kidney disease, which is a worldwide public health problem and presents a heavy burden to society. Hypoxia-inducible factor (HIF)-1α, the active subunit of HIF-1, has been reported to play a vital role in high phosphate-induced VC. However, the underlying mechanism is still undetermined, and effective treatment is unavailable. In the present study, human aortic smooth muscle cells (HASMCs) were cultured under normal or high phosphate media conditions. HIF-1α small interfering RNA and overexpression plasmids were employed to regulate HIF-1α expression. Phosphonoformic acid was employed to restrain the function of type III sodium-dependent phosphate cotransporter 1 (Pit-1). The expression levels of HIF-1α, Pit-1, runt-related transcription factor 2 (Runx2), and smooth muscle 22 alpha (SM22α) were evaluated, and the calcium contents were also examined. Cell growth was assessed using an MTT assay. High phosphate stimulation caused an upregulation in HIF-1α and Pit-1 expression levels and induced calcium depositions in HASMCs. Upregulation of Runx2 expression accompanied by downregulation of SM22α expression was observed in the high phosphate group. Following the suppression of HIF-1α expression, there was a concomitant attenuation in Pit-1 expression, calcium deposition, the alteration of phenotypic transition marker genes, and vice versa. The most serious calcium deposition was noted in HASMCs cultured under high phosphate conditions which were pretreated with a HIF-1α overexpression plasmid. However, when the biological functions of Pit-1 were restrained, the putative serious calcium deposition was not formed even in HASMCs transfected with a HIF-1α overexpression plasmid. The findings confirmed that HIF-1α regulated Pit-1 expression and exerted its pro-calcifying effect through Pit-1, which identified HIF-1α and Pit-1 as therapeutic targets for high phosphate-induced VC.

An update on clinical presentation and responses to therapy of patients with hereditary hypophosphatemic rickets with hypercalciuria (HHRH)

Pathogenic variants in solute carrier family 34, member 3 (SLC34A3), the gene encoding the sodium-dependent phosphate cotransporter 2c (NPT2c), cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH). Here, we report a pooled analysis of clinical and laboratory records of 304 individuals from 145 kindreds, including 20 previously unreported HHRH kindreds, in which two novel SLC34A3 pathogenic variants were identified. Compound heterozygous/homozygous carriers show above 90% penetrance for kidney and bone phenotypes. The biochemical phenotype for heterozygous carriers is intermediate with decreased serum phosphate, tubular reabsorption of phosphate (TRP (%)), fibroblast growth factor 23, and intact parathyroid hormone, but increased serum 1,25-dihydroxy vitamin D, and urine calcium excretion causing idiopathic hypercalciuria in 38%, with bone phenotypes still observed in 23% of patients. Oral phosphate supplementation is the current standard of care, which typically normalizes serum phosphate. However, although in more than half of individuals this therapy achieves correction of hypophosphatemia it fails to resolve the other outcomes. The American College of Medical Genetics score correlated with functional analysis of frequent SLC34A3 pathogenic variants in vitro and baseline disease severity. The number of mutant alleles and baseline TRP (%) were identified as predictors for kidney and bone phenotypes, baseline TRP (%) furthermore predicted response to therapy. Certain SLC34A3/NPT2c pathogenic variants can be identified with partial responses to therapy, whereas with some overlap, others present only with kidney phenotypes and a third group present only with bone phenotypes. Thus, our report highlights important novel clinical aspects of HHRH and heterozygous carriers, raises awareness to this rare group of disorders and can be a foundation for future studies urgently needed to guide therapy of HHRH.

Effect of an NHE3 inhibitor in combination with an NPT2b inhibitor on gastrointestinal phosphate absorption in Rodent models.

Many of the pathological consequences of chronic kidney disease can be attributed to an elevation in serum phosphate levels. Current therapies focused on decreasing intestinal phosphate absorption to treat hyperphosphatemia are inadequate. The most effective therapeutic strategy may be to target multiple absorptive pathways. In this study, the ability of a novel inhibitor of the intestinal sodium hydrogen exchanger 3 (NHE3), LY3304000, which inhibits paracellular, diffusional uptake of phosphate, to work in combination with an inhibitor of the active transporter, sodium dependent phosphate cotransporter 2b (NPT2b), LY3358966, was explored. LY3304000 modestly inhibited the acute uptake of phosphate into plasma of rats, while surprisingly, it doubled the rate of phosphate uptake in mice, an animal model dominated by NPT2b mediated acute phosphate uptake. In rats, LY3004000 and LY3358966 work in concert to inhibit acute phosphate uptake. On top of LY3358966, LY3304000 further decreased the acute uptake of phosphate into plasma. Studies measuring the recovery of radiolabeled phosphate in the intestine demonstrated LY3304000 and LY3358966 synergistically inhibited the absorption of phosphate in rats. We hypothesize the synergism is because the NHE3 inhibitor, LY3304000, has two opposing effects on intestinal phosphate absorption in rats, first it decreases diffusion mediated paracellular phosphate absorption, while second, it simultaneously increases phosphate absorption through the NPT2b pathway. NHE3 inhibition decreases proton export from enterocytes and raises the cell surface pH. In vitro, NPT2b mediated phosphate transport is increased at higher pHs. The increased NPT2b mediated transport induced by NHE3 inhibition is masked in rats which have relatively low levels of NPT2b mediated phosphate transport, by the more robust inhibition of diffusion mediated phosphate absorption. Thus, the inhibition of NPT2b mediated phosphate transport in rats in the presence of NHE3 inhibition has an effect that exceeds its effect in the absence of NHE3 inhibition, leading to the observed synergism on phosphate absorption between NPT2b and NHE3 inhibition.

Effects of the novel sodium-dependent phosphate cotransporter 2b inhibitor DZ1462 on hyperphosphatemia in chronic kidney disease.

Serum phosphate levels remain insufficiently controlled in chronic kidney disease (CKD) patients, and novel therapeutic strategies are needed. Blocking intestinal phosphate absorption mediated by sodium-dependent phosphate cotransporter type 2b (NPT2b) holds promise as one such strategy. The in vitro cellular potency of DZ1462 was evaluated using a radioactive Pi uptake assay on stable Chinese hamster ovary (CHO) cell clones transfected with human NPT2b (hNPT2b) or rat NPT2b (rNPT2b). The ability of DZ1462 to inhibit phosphate absorption was studied in vivo in an acute model after oral bolus challenge with 33PO4 and in an adenine-induced chronic hyperphosphatemia rat model. PK and minitox was also evaluated. The cellular assays with the hNPT2b-CHO and rNPT2b-CHO clones showed that DZ1462 significantly and potently inhibited phosphate uptake. In vivo, in a chronic Pi-fed rat model, DZ1462 effectively inhibited intestinal Pi uptake. In a hyperphosphatemia rat model, DZ1462 significantly inhibited Pi uptake, and DZ1462 in combination with sevelamer had a synergistic effect. The pharmacokinetics (PK) study confirmed that DZ1462 is a gastrointestinal (GI)-restricted compound that can remain in the intestine for a sufficient duration. In addition, DZ1462 also reduced cardiovascular events and ameliorated osteoporosis in a CKD animal model. This study revealed that a GI-restricted NPT2b inhibitor DZ1462 potently inhibits NPT2b in vitro and blocks intestinal phosphate uptake in multiple animal models with potential to reduce various cardiovascular events in CKD models. Therefore, DZ1462 may be useful to treat renal disease patients who have shown an unsatisfactory response to phosphate binders.

SGK3 promotes vascular calcification via Pit-1 in chronic kidney disease.

Rationale: Vascular calcification (VC) is a life-threatening complication in patients with chronic kidney disease (CKD) caused mainly by hyperphosphatemia. However, the regulation of VC remains unclear despite extensive research. Although serum- and glucocorticoid-induced kinase 3 (SGK3) regulate the sodium-dependent phosphate cotransporters in the intestine and kidney, its effect on VC in CKD remains unknown. Additionally, type III sodium-dependent phosphate cotransporter-1 (Pit-1) plays a significant role in VC development induced by high phosphate in vascular smooth muscle cells (VSMCs). However, it remains unclear whether SGK3 regulates Pit-1 and how exactly SGK3 promotes VC in CKD via Pit-1 at the molecular level. Thus, we investigated the role of SGK3 in the certified outflow vein of arteriovenous fistulas (AVF) and aortas of uremic mice. Methods and Results: In our study, using uremic mice, we observed a significant upregulation of SGK3 and calcium deposition in certified outflow veins of the AVF and aortas, and the increase expression of SGK3 was positively correlated with calcium deposition in uremic aortas. In vitro, the downregulation of SGK3 reversed VSMCs calcification and phenotype switching induced by high phosphate. Mechanistically, SGK3 activation enhanced the mRNA transcription of Pit-1 through NF-κB, downregulated the ubiquitin-proteasome mediated degradation of Pit-1 via inhibiting the activity of neural precursor cells expressing developmentally downregulated protein 4 subtype 2 (Nedd4-2), an E3 ubiquitin ligase. Moreover, under high phosphate stimulation, the enhanced phosphate uptake induced by SGK3 activation was independent of the increased protein expression of Pit-1. Our co-immunoprecipitation and in vitro kinase assays confirmed that SGK3 interacts with Pit-1 through Thr468 in loop7, leading to enhanced phosphate uptake. Conclusion: Thus, it is justifiable to conclude that SGK3 promotes VC in CKD by enhancing the expression and activities of Pit-1, which indicate that SGK3 could be a therapeutic target for VC in CKD.

Effects of dietary α-ketoglutarate on the growth performance, digestive enzymes, tor signaling pathway and intestinal microbiota of juvenile mirror carp (Cyprinus carpio) fed low phosphorus diets

The present study evaluated the effects of dietary α-ketoglutarate (AKG) on the growth performance, digestive enzymes, target of rapamycin (tor) signaling pathway and intestinal microbiota of juvenile mirror carp (Cyprinus carpio) fed low phosphorus diets. An eight week 2 × 3 two-factorial experiment was conducted to investigate the interaction between AKG levels at 0 or 0.6%, and dietary phosphorus levels at 0.27%, 0.55% or 0.76%. The results showed that the final body weight (FBW) and weight growth rate (WGR) were significantly improved and the feed conversion ratio (FCR) was significantly decreased by the dietary AKG or phosphorus (P < 0.05). The crude ash and phosphorous content were significantly increased with increasing available phosphorus supplement from 0.27% to 0.76%, whereas crude lipid was decreased as dietary phosphorus levels increased (P < 0.05). Fish fed a diet supplemented with 0.6% AKG showed a significant increase in crude protein, crude ash and phosphorous content compared to the diet without AKG group (P < 0.05). The mRNA expression of IIb sodium-dependent phosphate cotransporter (NaPi-IIb) was decreased by dietary AKG or phosphorus (P < 0.05).The activities of trypsin was significantly improved by dietary AKG or phosphorus (P < 0.05). The mRNA expression of tor, insulin growth factor I (igfI) and growth hormone (gh) were significantly increased by dietary AKG or phosphorus (P < 0.05). The richness estimate of Ace and Chao, and the diversity estimators of Simpson were significantly decreased with increasing phosphorus levels from 0.27% to 0.55% (P < 0.05). Compared with the 0.27% phosphorus group, the abundance of Fusobacteriota and Bacteroidota were relatively increased and the abundance of Proteobacteria, Actinobacteriota and Cyanobacteria were relatively decreased in 0.27% phosphorus added 0.6% AKG group. The abundance of Fusobacteriota and Actinobacteriota in 0.27% phosphorus group was relatively lower than that in 0.55% phosphorus group. On the contrary, the abundance of Firmicutes, Bacteroidota and Cyanobacteria in 0.27% phosphorus group was relatively higher than that in 0.55% phosphorus groups. These results indicated that dietary supplementation with 0.6% AKG could improve growth performance by increasing the activities of trypsin and promoting the formation of beneficial intestinal microbiota, and the tor, gh/igf-I gene expression in liver of mirror carp fed low phosphorus diets.

Kidney glycolysis serves as a mammalian phosphate sensor that maintains phosphate homeostasis.

How phosphate levels are detected in mammals is unknown. The bone-derived hormone fibroblast growth factor 23 (FGF23) lowers blood phosphate levels by reducing kidney phosphate reabsorption and 1,25(OH)2D production, but phosphate does not directly stimulate bone FGF23 expression. Using PET scanning and LC-MS, we found that phosphate increases kidney-specific glycolysis and synthesis of glycerol-3-phosphate (G-3-P), which then circulates to bone to trigger FGF23 production. Further, we found that G-3-P dehydrogenase 1 (Gpd1), a cytosolic enzyme that synthesizes G-3-P and oxidizes NADH to NAD+, is required for phosphate-stimulated G-3-P and FGF23 production and prevention of hyperphosphatemia. In proximal tubule cells, we found that phosphate availability is substrate-limiting for glycolysis and G-3-P production and that increased glycolysis and Gpd1 activity are coupled through cytosolic NAD+ recycling. Finally, we show that the type II sodium-dependent phosphate cotransporter Npt2a, which is primarily expressed in the proximal tubule, conferred kidney specificity to phosphate-stimulated G-3-P production. Importantly, exogenous G-3-P stimulated FGF23 production when Npt2a or Gpd1 were absent, confirming that it was the key circulating factor downstream of glycolytic phosphate sensing in the kidney. Together, these findings place glycolysis at the nexus of mineral and energy metabolism and identify a kidney-bone feedback loop that controls phosphate homeostasis.

PS-B01-11: GENERATION OF TRANSGENIC MICE WITH PROXIMAL TUBULE-SPECIFIC OVEREXPRESSION OF ATRAP AND ANALYSIS OF PROXIMAL TUBULE ATRAP IN BLOOD PRESSURE REGULATION

Objective: Proximal tubule angiotensin II type 1 receptor (AT1R) is involved in blood pressure (BP) regulation (Li XC, et al. Hypertension, 77:1285-1298, 2021). AT1 receptor-associated protein (ATRAP) interacts with AT1R and promotes constitutive internalization of AT1R, to inhibit pathological activation of its downstream signaling (Tamura K, et al. Hypertens Res, 45:32–39, 2022). Endogenous ATRAP is abundantly expressed along kidney tubules in the kidney, and our previous investigations using systemic ATRAP knockout and distal tubule-dominant ATRAP transgenic mice revealed that ATRAP suppressed Ang II-induced hypertension mainly through a distal tubule-mediated mechanism (Wakui H, et al. Hypertension, 61:1203–10, 2013; Ohsawa M, et al. Kidney Int, 86:570–81, 2014). On the other hand, proximal tubule-specific ATRAP knockout mice exhibited similar baseline BP and pressor response to Ang II compared with wild-type mice (Kinguchi S, et al. J Am Heart Assoc, 8(8):e012395, 2019). The present study was performed to investigate the effects of enhancement of proximal tubule ATRAP on Ang II-mediated hypertension, using proximal tubule-specific ATRAP transgenic mice. Design and method: [Experiment 1] Using mouse ATRAP cDNA linked to the type II sodium-dependent phosphate cotransporters (NPT2) promoter, we generated transgenic mice (NPT2-Tg mice) with proximal tubule-specific overexpression of ATRAP. ATRAP mRNA expression in the proximal tubule was quantified by a laser capture microdissection and quantitative reverse transcription-polymerase chain reaction analysis. [Experiment 2] Male NPT2-Tg and wild-type littermate control (LC) mice were divided into Ang II (1000 ng/kg/min) and vehicle groups. BP was measured using a tail-cuff method before and 14 days after treatment, and then mice were sacrificed and their organs were collected. Results: [Experiment 1] 5 of 12 lines of ATRAP Tg mice exhibited kidney overexpression of the ATRAP-transgene in comparison with LC mice. Kidney ATRAP expression at the protein level revealed the highest expression level of ATRAP in lines 23, approximately 10-fold higher compared to LC mice. Therefore, we chose line 23 (NPT2-Tg23) for further analysis. ATRAP mRNA expression was increased approximately 9-fold in the proximal tubules of NPT2-Tg23 mice compared with LC mice. On the other hand, ATRAP mRNA expression in the distal tubules was comparable between NPT2-Tg23 and LC mice. [Experiment 2] We are currently conducting the experiments and will present our findings at this conference. Conclusions: We succeeded in generating transgenic mice with proximal tubule-specific overexpression of ATRAP. We plan to present the results of the impact of enhancement of proximal tubular ATRAP on Ang II-dependent hypertension at this conference.

Binding and inhibitory activities: A novel oral therapeutic agent for the treatment of hyperphosphataemia rats

Novel oral therapeutic agents based on inhibition or binding activity without adverse events in CKD patients are urgently needed. Here, 5/6 nephrectomy (NX) rats were used to construct a CKD model. Aminated cellulose (AC711), which is metal-free, non-absorbable, and low-volume expansive, was used as a novel oral therapeutic agent for hyperphosphataemia treatment in rats. The efficacy of AC711 on serum and urinary phosphate levels, the expression of type II sodium-dependent phosphate cotransporter (NPT2b), and type III Na-dependent phosphate cotransporter (PiT-1/2) was examined. Serum fibroblast growth factor-23 (FGF-23) levels, parathyroid hormone (PTH) levels, and the phenotypic transformation of vascular smooth muscle cell markers (smooth muscle 22 (SM22) and Runx2) are considered an adaptive response to elevated serum phosphate levels. A similar efficacy of AC711 was observed on serum and urinary phosphate levels when the same dose of AC711 and sevelamer was administered to 5/6 NX rats. The decreasing expression of NPT2b, PiT-1, and PiT-2 was examined in the AC711 groups in a dose-dependent manner. The sevelamer and AC711-MD groups for FGF-23 and PTH indicated no significant difference. The down-regulation of Runx2 expression and up-regulation of SM22 expression were seen in the AC711 groups in a dose-dependent manner. Two suppression mechanisms (binding and inhibiting activities) were observed in the gastrointestinal (GI) tract in the AC711 groups. A novel oral phosphate binder, AC711, showed both binding and inhibition characteristics. The low-volume expansion of AC711 following exposure to simulated intestinal fluid provides the potential therapeutic benefits with the advantage of moderate GI side effects.

Effect of the gene silencing of phosphorus transporters on phosphorus absorption across primary cultured duodenal epithelial cell monolayers of chick embryos

The aim of the study was to investigate whether phosphorus (P) transporters, type IIb sodium-dependent phosphate cotransporter (NaP-IIb) and inorganic phosphate transporter 2 (PiT2), were directly involved in P absorption across primary cultured duodenal epithelial cell monolayers of chick embryos. The siRNAs against NaP-IIb or PiT2 were designed, synthesized and transfected into primary cultured duodenal epithelial cells of chick embryos. Then, the inhibitory efficiency of siRNAs against NaP-IIb or PiT2 was analyzed, and the most efficacious siRNAs were selected to be used for subsequent P absorption experiments. Briefly, primary cultured duodenal epithelial cells of chick embryos were transfected with either NaP-IIb or PiT2 siRNAs and grown in confluent monolayers on transwell plates. The untransfected or transfected cell monolayers were then incubated in an uptake medium containing 0 or 0.25 mmol L–1 of P as KH2PO4 to measure the P absorption across duodenal epithelial cell monolayers. The results showed that among the siRNAs designed, si-1372 and si-890 were demonstrated to be the most effective in inhibiting the NaP-IIb and PiT2 expressions, respectively. Supplemental P increased (P=0.065) the protein abundance of PiT2 and enhanced (P<0.0001) P absorption in primary cultured duodenal epithelial cell of chick embryos. Furthermore, NaP-IIb silencing decreased (P=0.07) P absorption across duodenal epithelial cell monolayers, while PiT2 silencing had no effect (P=0.345). It is concluded that the NaP-IIb, but not PiT2, might be directly involved in the P absorption of chick duodenal epithelial cells.

Review of Knowledge of Uranium-Induced Kidney Toxicity for the Development of an Adverse Outcome Pathway to Renal Impairment

An adverse outcome pathway (AOP) is a conceptual construct of causally and sequentially linked events, which occur during exposure to stressors, with an adverse outcome relevant to risk assessment. The development of an AOP is a means of identifying knowledge gaps in order to prioritize research assessing the health risks associated with exposure to physical or chemical stressors. In this paper, a review of knowledge was proposed, examining experimental and epidemiological data, in order to identify relevant key events and potential key event relationships in an AOP for renal impairment, relevant to stressors such as uranium (U). Other stressors may promote similar pathways, and this review is a necessary step to compare and combine knowledge reported for nephrotoxicants. U metal ions are filtered through the glomerular membrane of the kidneys, then concentrate in the cortical and juxtaglomerular areas, and bind to the brush border membrane of the proximal convoluted tubules. U uptake by epithelial cells occurs through endocytosis and the sodium-dependent phosphate co-transporter (NaPi-IIa). The identified key events start with the inhibition of the mitochondria electron transfer chain and the collapse of mitochondrial membrane potential, due to cytochrome b5/cytochrome c disruption. In the nucleus, U directly interacts with negatively charged DNA phosphate, thereby inducing an adduct formation, and possibly DNA strand breaks or cross-links. U also compromises DNA repair by inhibiting zing finger proteins. Thereafter, U triggers the Nrf2, NF-κB, or endoplasmic reticulum stress pathways. The resulting cellular key events include oxidative stress, DNA strand breaks and chromosomal aberrations, apoptosis, and pro-inflammatory effects. Finally, the main adverse outcome is tubular damage of the S2 and S3 segments of the kidneys, leading to tubular cell death, and then kidney failure. The attribution of renal carcinogenesis due to U is controversial, and specific experimental or epidemiological studies must be conducted. A tentative construction of an AOP for uranium-induced kidney toxicity and failure was proposed.

Effects of pharmacological inhibition of the sodium-dependent phosphate cotransporter 2b (NPT2b) on intestinal phosphate absorption in mouse and rat models.

An excess phosphate burden in renal disease has pathological consequences for bone, kidney, and heart. Therapies to decrease intestinal phosphate absorption have been used to address the problem, but with limited success. Here, we describe the in vivo effects of a novel potent inhibitor of the intestinal sodium‐dependent phosphate cotransporter NPT2b, LY3358966. Following treatment with LY3358966, phosphate uptake into plasma 15 min following an oral dose of radiolabeled phosphate was decreased 74% and 22% in mice and rats, respectively, indicating NPT2b plays a much more dominant role in mice than rats. Following the treatment with LY3358966 and radiolabeled phosphate, mouse feces were collected for 48 h to determine the ability of LY3358966 to inhibit phosphate absorption. Compared to vehicle‐treated animals, there was a significant increase in radiolabeled phosphate recovered in feces (8.6% of the dose, p < .0001). Similar studies performed in rats also increased phosphate recovered in feces (5.3% of the dose, p < .05). When used in combination with the phosphate binder sevelamer in rats, there was a further small, but not significant, increase in fecal phosphate. In conclusion, LY3358966 revealed a more prominent role for NPT2b on acute intestinal phosphate uptake into plasma in mice than rats. However, the modest effects on total intestinal phosphate absorption observed in mice and rats with LY3359866 when used alone or in combination with sevelamer highlights the challenge to identify new more effective therapeutic targets and/or drug combinations to treat the phosphate burden in patients with renal disease.

Abstract 10574: Living with Sexual Partner Slowed Klotho Deficiency-Induced Heart Aging via Suppressing Renal Sodium-Phosphate Co-Transporters

Background: Staying young at heart is a dream of many aged people. Sexuality is now generally considered beneficial for the overall health. It reduces the risk of a heart attack. However, while the concept of “sex improves health” has been demonstrated, the underlying mechanism remains poorly understood. Methods &amp; Results: Klotho mutant homozygous (KL-/-) mice and age- and sex-matched wild-type (WT) mice were used. One male and one female mouse were co-housed from weaning and fed with low-phosphorus diet to reduce serum phosphate level. And the mice co-housing with the same gender were used as control. Body weight was measured weekly and lifespan was recorded. Heart function was measured by magnetic resonance imaging (MRI). Mice were sacrificed just before dying. Klotho-deficient mice demonstrated heart aging phenotype, as evidenced by cardiac remodeling and cardiac dysfunction. The compromised cardiac function was associated with increased oxidative stress, mitochondrial damage and myocyte apoptosis. We showed that hyperphosphatemia contributes to Klotho deficiency-induced cardiac damages. Interestingly, sexuality attenuated cardiac aging. Mechanistically, sex increased estrogen secretion, which decreased hyperphosphatemia through inhibition of renal tubular sodium-dependent phosphate (NaPi) co-transporters. Co-housing sexual partners also attenuated oxidative stress and myocyte apoptosis and improved mitochondrial function. Moreover, living with sexual partners extends the lifespan by 86.8% in KL(-/-) female mice and 38.7% in male mice. Conclusion: In the Klotho deficiency-induced aging model, sexuality improves heart aging and extends lifespan. This study demonstrated for the first time that living with sexual partners increases estrogen secretion which inhibits renal NaPi co-transporters leading to attenuation Klotho deficiency-induced hyperphosphatemia and heart aging.

Phosphate Overload Stimulates Inflammatory Reaction via PiT-1 and Induces Vascular Calcification in Uremia

Vascular calcification (VC) is an important risk factor for cardiovascular disease in maintenance hemodialysis (MHD) patients. Hyperphosphatemia and microinflammation statement are known major contributors to the development of VC; however, the mechanisms are unknown. The aims of this study were to explore the risk factors of VC in MHD patients and to explore whether high phosphate could increase the secretion of inflammatory cytokines via PiT-1 in monocytes. A cross-sectional study was conducted on 65 MHD patients to assess the relevance of coronary artery calcification (CAC), inflammatory factors, serum phosphate, and sodium-dependent phosphate cotransporter (NPT) mRNA expression of peripheral blood mononuclear cells (PBMCs). Multivariate logistic regression analysis was used to analyze the predictors of CAC. The calcification effects of high phosphate (HP), TNF-α, and supernatants of healthy human monocytes treated with HP were further evaluated in cultured HASMCs. Diabetes, longer dialysis vintage, higher serum TNF-α levels, and PiT-1 mRNA expression of PBMCs) were independent risk factors of CAC in MHD patients. The mRNA levels of PiT-1 in PBMCs were positively correlated with serum phosphate, CAC scores, and Pit-2 mRNA levels of PBMCs. The expressions of TNF-α, IL-6, and PiT-1 in human monocytes were significantly increased in a dose-dependent manner after treatment with HP, which was subsequently inhibited by NPT antagonist phosphonoformic acid. Neither TNF-α alone nor supernatants of monocytes stimulated with HP promoted the expression of osteopontin and Runt-related transcription factor 2 (Runx2) or caused mineralization in human aortic smooth muscle cells, but combined with HP intervention, the calcification effects were markedly increased in human aortic smooth muscle cells and ameliorated by phosphonoformic acid treatment. Hyperphosphatemia directly increased the synthesis and secretion of TNF-α by monocytes may via PiT-1 pathway, resulting in elevated systemic inflammatory response, which may further aggravate VC induced by phosphate overload in MHD patients.

Mechanisms of Epidermal Growth Factor Effect on Animal Intestinal Phosphate Absorption: A Review.

Phosphorus is one of the essential mineral elements of animals that plays an important role in animal growth and development, bone formation, energy metabolism, nucleic acid synthesis, cell signal transduction, and blood acid–base balance. It has been established that the Type IIb sodium-dependent phosphate cotransporters (NaPi-IIb) protein is the major sodium-dependent phosphate (Pi) transporter, which plays an important role in Pi uptake across the apical membrane of epithelial cells in the small intestine. Previous studies have demonstrated that epidermal growth factor (EGF) is involved in regulating intestinal Pi absorption. Here we summarize the effects of EGF on active Pi transport of NaPi-IIb under different conditions. Under normal conditions, EGF inhibits the active transport of Pi by inhibiting the expression of NaPi-IIb, while, under intestinal injury condition, EGF promotes the active absorption of Pi through upregulating the expression of NaPi-IIb. This review provides a reference for information about EGF-regulatory functions in Pi absorption in the animal intestine.

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

Abstract 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.

FGF23 functions and disease.

The main function of fibroblast growth factor 23 (FGF23) is the regulation of phosphate metabolism through its action on the sodium-dependent phosphate cotransporters in the proximal renal tubules. Additionally, FGF23 interacts with vitamin D and parathyroid hormone in a complex metabolic pathway whose detailed mechanisms are still not clear in human physiology and disease. More recently, research has also focused on the understanding of mechanisms of FGF23 action on organs and system other than the kidneys and bone, as well as on its interaction with other metabolic pathways. Collectively, the new evidence are successfully used for the clinical evaluation and management of FGF23-related disorders, for which new therapies with many potential applications are now available.

Renal Dnase1 expression is regulated by FGF23 but loss of Dnase1 does not alter renal phosphate handling

Fibroblast growth factor 23 (FGF23) is a bone-derived endocrine hormone that regulates phosphate and vitamin D metabolism. In models of FGF23 excess, renal deoxyribonuclease 1 (Dnase1) mRNA expression is downregulated. Dnase-1 is an endonuclease which binds monomeric actin. We investigated whether FGF23 suppresses renal Dnase-1 expression to facilitate endocytic retrieval of renal sodium dependent phosphate co-transporters (NaPi-IIa/c) from the brush border membrane by promoting actin polymerization. We showed that wild type mice on low phosphate diet and Fgf23−/− mice with hyperphosphatemia have increased renal Dnase1 mRNA expression while in Hyp mice with FGF23 excess and hypophosphatemia, Dnase1 mRNA expression is decreased. Administration of FGF23 in wild type and Fgf23−/− mice lowered Dnase1 expression. Taken together, our data shows that Dnase1 is regulated by FGF23. In 6-week-old Dnase1−/− mice, plasma phosphate and renal NaPi-IIa protein were significantly lower compared to wild-type mice. However, these changes were transient, normalized by 12 weeks of age and had no impact on bone morphology. Adaptation to low and high phosphate diet were similar in Dnase1−/− and Dnase1+/+ mice, and loss of Dnase1 gene expression did not rescue hyperphosphatemia in Fgf23−/− mice. We conclude that Dnase-1 does not mediate FGF23-induced inhibition of renal tubular phosphate reabsorption.

Noncanonical Sequences Involving NHERF1 Interaction with NPT2A Govern Hormone-Regulated Phosphate Transport: Binding Outside the Box.

Na+/H+ exchange factor-1 (NHERF1), a multidomain PDZ scaffolding phosphoprotein, is required for the type II sodium-dependent phosphate cotransporter (NPT2A)-mediated renal phosphate absorption. Both PDZ1 and PDZ2 domains are involved in NPT2A-dependent phosphate uptake. Though harboring identical core-binding motifs, PDZ1 and PDZ2 play entirely different roles in hormone-regulated phosphate transport. PDZ1 is required for the interaction with the C-terminal PDZ-binding sequence of NPT2A (-TRL). Remarkably, phosphocycling at Ser290 distant from PDZ1, the penultimate step for both parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) regulation, controls the association between NHERF1 and NPT2A. PDZ2 interacts with the C-terminal PDZ-recognition motif (-TRL) of G Protein-coupled Receptor Kinase 6A (GRK6A), and that promotes phosphorylation of Ser290. The compelling biological puzzle is how PDZ1 and PDZ2 with identical GYGF core-binding motifs specifically recognize distinct binding partners. Binding determinants distinct from the canonical PDZ-ligand interactions and located “outside the box” explain PDZ domain specificity. Phosphorylation of NHERF1 by diverse kinases and associated conformational changes in NHERF1 add more complexity to PDZ-binding diversity.

Multisite NHERF1 phosphorylation controls GRK6A regulation of hormone-sensitive phosphate transport

The type II sodium-dependent phosphate cotransporter (NPT2A) mediates renal phosphate uptake. The NPT2A is regulated by parathyroid hormone (PTH) and fibroblast growth factor 23, which requires Na+/H+ exchange regulatory factor-1 (NHERF1), a multidomain PDZ-containing phosphoprotein. Phosphocycling controls the association between NHERF1 and the NPT2A. Here, we characterize the critical involvement of G protein–coupled receptor kinase 6A (GRK6A) in mediating PTH-sensitive phosphate transport by targeted phosphorylation coupled with NHERF1 conformational rearrangement, which in turn allows phosphorylation at a secondary site. GRK6A, through its carboxy-terminal PDZ recognition motif, binds NHERF1 PDZ1 with greater affinity than PDZ2. However, the association between NHERF1 PDZ2 and GRK6A is necessary for PTH action. Ser162, a PKCα phosphorylation site in PDZ2, regulates the binding affinity between PDZ2 and GRK6A. Substitution of Ser162 with alanine (S162A) blocks the PTH action but does not disrupt the interaction between NHERF1 and the NPT2A. Replacement of Ser162 with aspartic acid (S162D) abrogates the interaction between NHERF1 and the NPT2A and concurrently PTH action. We used amber codon suppression to generate a phosphorylated Ser162(pSer162)-PDZ2 variant. KD values determined by fluorescence anisotropy indicate that incorporation of pSer162 increased the binding affinity to the carboxy terminus of GRK6A 2-fold compared with WT PDZ2. Molecular dynamics simulations predict formation of an electrostatic network between pSer162 and Asp183 of PDZ2 and Arg at position −1 of the GRK6A PDZ-binding motif. Our results suggest that PDZ2 plays a regulatory role in PTH-sensitive NPT2A-mediated phosphate transport and phosphorylation of Ser162 in PDZ2 modulates the interaction with GRK6A.