Renal Pi Research Articles

Controlled dietary phosphate loading in healthy young men elevates plasma phosphate and FGF23 levels.

Increased dietary inorganic phosphate (Pi) intake stimulates renal Pi excretion, in part, by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23) or dopamine. High dietary Pi may also stimulate sympathetic outflow. Rodent studies provided evidence for these regulatory loops, while controlled experiments in healthy humans examined periods of either a few hours or several weeks, and often varied dietary calcium intake. The effects of controlled, isolated changes in dietary Pi intake over shorter periods are unknown. We studied the effects of a low or high Pi diet on parameters of mineral metabolism in 10 healthy young men. Participants received a standardized diet (1000mg phosphorus equivalent/day) supplemented with either a phosphate binder (low Pi diet) or phosphate capsules (750mg phosphorus, high Pi diet) in a randomized cross-over trial for 5days with a 7-day washout between diets. High Pi intake increased plasma Pi levels and 24-h excretion and decreased urinary calcium excretion. High Pi intake increased intact FGF23 (iFGF23) and suppressed plasma Klotho without affecting cFGF23, PTH, calcidiol, calcitriol, Fetuin-A, dopamine, epinephrine, norepinephrine, metanephrine, or aldosterone. Higher iFGF23 correlated with lower calcitriol and higher PTH. These data support a role for iFGF23 in increasing renal Pi excretion and reducing calcitriol in healthy young men during steady-state high dietary Pi intake. High dietary Pi intake elevated blood Pi levels in healthy young subjects with normal renal function and may therefore be a health risk, as higher serum Pi levels are associated with cardiovascular risk in the general population.

Role of Leptin in Regulating Renal Phosphate Transport

Elevated plasma phosphate (Pi) levels, known as hyperphosphatemia, are associated with an increased risk of cardiovascular complications and mortality. Although studies have suggested a correlation between obesity and hyperphosphatemia/phosphate toxicity, the root cause of this observation has not been established. Since hyperleptinemia was also found to be associated with abnormalities in bone structure, we hypothesized that effects on renal Pi transport are responsible for this observation. To address this question, we studied hyperleptinemic mice (db/db, n=14) and compared them to wild-type mice (WT, n=10). Blood and urine samples, as well as kidney tissues, were collected and analyzed. Body weight was approximately twice as high in db/db compared to WT mice (59±3 vs. 30±2 g, P<0.05). Plasma Pi levels in db/db mice were significantly greater (~1.5-fold) compared to WT mice (2.6±0.1 vs. 1.7±0.1 mmol/L, P<0.05). However, urinary Pi /creatinine ratios were similar between genotypes. Plasma Ca2+ levels in db/db mice were significantly higher compared to WT mice (2.68±0.03 vs. 2.48±0.03 mmol/L, P<0.05) alongside urinary Ca2+/creatinine ratios being significantly higher in db/db mice compared to WT mice (1.6±0.4 vs. 0.3±0.1 mmol/mmol, P<0.05). Plasma parathyroid hormone (PTH, regulating Pi and Ca2+) levels were similar in both genotypes. The phosphaturic hormone fibroblast growth factor 23 (FGF23), which is produced and released from bone, was slightly but significantly elevated in db/db compared to WT mice (254±12 vs. 227±18 pg/mL, P<0.05). Bone formation markers (osteocalcin and procollagen type I N-propeptide) and bone resorption markers (tartrate-resistant acid phosphatase 5b and C-terminal telopeptide of type I collagen) were similar between genotypes, indicating that Pi and Ca2+ release from bone may not be causing these mineral differences. To determine the role of the kidney for the development of hyperphosphatemia, we performed 32P radiotracer studies in acutely isolated renal brush border membrane vesicles. Sodium-dependent 32P transport was not significantly different between genotypes. To determine the ambient in vivo contribution of the Na+-Pi cotransporter Npt2a, we employed acute pharmacological inhibition with PF-06869206 (Npt2a inhibitor, 30 mg/kg b.w, n=7) or vehicle (n=11) and compared plasma Pi before and 2 hours after administration in db/db mice. Vehicle treatment slightly increased plasma Pi levels compared to baseline (2.6±0.1 vs. 2.1±0.1 mmol/L, P<0.05); in contrast, the Npt2a inhibitor significantly reduced (~50%) plasma Pi levels compared to baseline (1.3±0.1 vs. 2.5±0.1 mmol/L, P<0.05). In summary, our study demonstrates that hyperphosphatemia in db/db mice is not caused by changes in renal Pi transport or bone turnover, suggesting that possibly intestinal mechanisms are responsible for increased Pi, and possibly Ca2+, absorption. Of note, Npt2a inhibition was effective in reducing hyperphosphatemia in db/db mice, indicating a potential therapeutic strategy. This work was supported by a VA Merit Review Award IBX004968A (to Dr. Rieg) and a Pilot Project from the USF Microbiomes Institute (to T.R. and J.D.R). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The calcium-sensing receptor has only a parathyroid hormone-dependent role in the acute response of renal phosphate transporters to phosphate intake.

The kidney controls systemic inorganic phosphate (Pi) levels by adapting reabsorption to Pi intake. Renal Pi reabsorption is mostly mediated by sodium-phosphate cotransporters NaPi-IIa (SLC34A1) and NaPi-IIc (SLC34A3) that are tightly controlled by various hormones including parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23). PTH and FGF23 rise in response to Pi intake and decrease NaPi-IIa and NaPi-IIc brush border membrane abundance enhancing phosphaturia. Phosphaturia and transporter regulation occurs even in the absence of PTH and FGF23 signaling. The calcium-sensing receptor (CaSR) regulates PTH and FGF23 secretion, and may also directly affect renal Pi handling. Here, we combined pharmacological and genetic approaches to examine the role of the CaSR in the acute phosphaturic response to Pi loading. Animals pretreated with the calcimimetic cinacalcet were hyperphosphatemic, had blunted PTH levels upon Pi administration, a reduced Pi-induced phosphaturia, and no Pi-induced NaPi-IIa downregulation. The calcilytic NPS-2143 exaggerated the PTH response to Pi loading but did not abolish Pi-induced downregulation of NaPi-IIa. In mice with a dominant inactivating mutation in the Casr (CasrBCH002), baseline NaPi-IIa expression was higher, whereas downregulation of transporter expression was blunted in double CasrBCH002/PTH knockout (KO) transgenic animals. Thus, in response to an acute Pi load, acute modulation of the CaSR affects the endocrine and renal response, whereas chronic genetic inactivation, displays only subtle differences in the downregulation of NaPi-IIa and NaPi-IIc renal expression. We did not find evidence that the CaSR impacts on the acute renal response to oral Pi loading beyond its role in regulating PTH secretion.NEW & NOTEWORTHY Consumption of phosphate-rich diets causes an adaptive response of the body leading to the urinary excretion of phosphate. The underlying mechanisms are still poorly understood. Here, we examined the role of the calcium-sensing receptor (CaSR) that senses both calcium and phosphate. We confirmed that the receptor increases the secretion of parathyroid hormone involved in stimulating urinary phosphate excretion. However, we did not find any evidence for a role of the receptor beyond this function.

FGF23 directly inhibits osteoprogenitor differentiation in Dmp1-knockout mice.

Fibroblast growth factor 23 (FGF23) is a phosphate-regulating (Pi-regulating) hormone produced by bone. Hereditary hypophosphatemic disorders are associated with FGF23 excess, impaired skeletal growth, and osteomalacia. Blocking FGF23 became an effective therapeutic strategy in X-linked hypophosphatemia, but testing remains limited in autosomal recessive hypophosphatemic rickets (ARHR). This study investigates the effects of Pi repletion and bone-specific deletion of Fgf23 on bone and mineral metabolism in the dentin matrix protein 1-knockout (Dmp1KO) mouse model of ARHR. At 12 weeks, Dmp1KO mice showed increased serum FGF23 and parathyroid hormone levels, hypophosphatemia, impaired growth, rickets, and osteomalacia. Six weeks of dietary Pi supplementation exacerbated FGF23 production, hyperparathyroidism, renal Pi excretion, and osteomalacia. In contrast, osteocyte-specific deletion of Fgf23 resulted in a partial correction of FGF23 excess, which was sufficient to fully restore serum Pi levels but only partially corrected the bone phenotype. In vitro, we show that FGF23 directly impaired osteoprogenitors' differentiation and that DMP1 deficiency contributed to impaired mineralization independent of FGF23 or Pi levels. In conclusion, FGF23-induced hypophosphatemia is only partially responsible for the bone defects observed in Dmp1KO mice. Our data suggest that combined DMP1 repletion and FGF23 blockade could effectively correct ARHR-associated mineral and bone disorders.

Comparative assessment of renal volume and doppler velocimetric indices among subjects with sickle cell disease and controls in Benin, Nigeria

Background: Sickle Cell Disease (SCD), a hereditary blood disorder caused by an abnormality in haemoglobin can be complicated by impairment of renal function. Renal Doppler ultrasound has been found to be an effective method of evaluating reno-vascular events prior to abnormal laboratory renal function tests.
 Aim and Objectives: This study aimed to evaluate and compare the renal volume (RV), intra-renal resistive and pulsatility indices (RI, PI) among sickle cell patients and controls in UBTH, Benin City using ultrasonography.
 Materials and Method: This was a cross-sectional comparative study of renal volume, intra-renal resistive and pulsatility indices among 50 sickle cell disease patients attending sickle cell clinic of the University of Benin Teaching Hospital and equal number of “Age and Sex” matched controls. The study was conducted using a 2-8MHz curvilinear transducer of a SONOACE X6 (Medison Inc., Korea 2010) Doppler ultrasound machine.
 Data analysis: Collated data was analysed using IBM SPSS Statistics for Windows, Version 23.0 (IBM Corp, Armonk. N.Y., USA)
 Results: The mean age for the HbSS subjects was 22.4±3.7years while that of the control group was 23.3 ±4.5 years. The left RV was higher than the right RV in HbSS and HbAA: 169.3±40.2 cm3 versus 162.2±40.3 cm3 and 153.9±30.9cm3 versus 134.7±26.4 cm3 respectively. The mean RV, RI and PI was significantly higher in HbSS than controls (RV: 165.8±39.8cm versus 122.9±13.4 cm3 ; p = 0.0001, RI: 0.74±0.02 versus 0.61±0.04; p= 0.0001, PI: 1.43±0.06 cm3 versus 0.90±0.05; p = 0.0001).
 Conclusion: Renal volume, RI and PI were statistically significantly higher in HbSS patients than controls.

Fetal Renal Artery Doppler in Cases of Isolated Oligohydramnios: Can it Predict Adverse Neonatal Outcome?

Aims and Objectives: This study compares the fetal renal artery flow velocity waveforms of pregnancies complicated by oligohydramnios with normal pregnancies and to detect any statistically significant difference between the two Groups and to further determine the relationship of these findings with neonatal outcome. Method: All pregnant women in IIIrd trimester with gestational age between 32 weeks to 36 weeks with isolated oligohydramnios were included as cases (Group-A). Normal IIIrd trimester pregnancies (Group-B) with gestational age between 32 weeks to 36 weeks served as controls between the study period of Jan 2018 to Dec 2019. Results: The mean umbilical artery pulsatility index (PI; mean ± SD) in patients of Group-A was 1.0730 ± 0.0637 and that of patients in Group-B was 1.0012 ± 0.1400 with P = 0.0023. The mean umbilical artery S/D ratio (mean ± SD) in patients of Group-A, was 2.9120 ± 0.2476 and that of patients in Group-B was 2.5135 ± 0.2751 with P < 0.0001. The mean renal artery PI (mean ± SD) in patients of Group-A was 2.5863 ± 0.2608 and that of patients in Group-B was 2.4737 ± 0.2648 with P = 0.0240. The mean birth weight and Apgar Score were also found to be statistically significant between the two Groups. Conclusion: The distribution of mean renal artery PI and umbilical artery PI, in oligohydramnios cases, was higher in comparison to normal cases. As the Neonatal intensive care unit (NICU) admission was higher in the oligohydramnios group with abnormal renal artery doppler indices, our recommendation is that abnormal fetal renal artery Doppler indices are an indication for early referral to tertiary care setup, as the majority of these neonates will require NICU care postdelivery.

Mild Chronic Kidney Disease Associated with Low Bone Formation and Decrease in Phosphate Transporters and Signaling Pathways Gene Expression.

The initial phases of molecular and cellular maladaptive bone responses in early chronic kidney disease (CKD) remain mostly unknown. We induced mild CKD in spontaneously hypertensive rats (SHR) by either causing arterial hypertension lasting six months (sham-operated rats, SO6) or in its' combination with 3/4 nephrectomy lasting two and six months (Nx2 and Nx6, respectively). Sham-operated SHRs (SO2) and Wistar Kyoto rats (WKY2) with a two-month follow-up served as controls. Animals were fed standard chow containing 0.6% phosphate. Upon follow-up completion in each animal, we measured creatinine clearance, urine albumin-to-creatinine ratio, renal interstitial fibrosis, inorganic phosphate (Pi) exchange, intact parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), Klotho, Dickkopf-1, sclerostin, and assessed bone response by static histomorphometry and gene expression profiles. The mild CKD groups had no increase in renal Pi excretion, FGF23, or PTH levels. Serum Pi, Dickkopf-1, and sclerostin were higher in Nx6. A decrease in trabecular bone area and osteocyte number was obvious in SO6. Nx2 and Nx6 had additionally lower osteoblast numbers. The decline in eroded perimeter, a resorption index, was only apparent in Nx6. Significant downregulation of genes related to Pi transport, MAPK, WNT, and BMP signaling accompanied histological alterations in Nx2 and Nx6. We found an association between mild CKD and histological and molecular features suggesting lower bone turnover, which occurred at normal levels of systemic Pi-regulating factors.

Doppler velocity of fetal renal arteries in case of oligohydramnios

Background: Oligohydramnios is a relatively common complication of pregnancy and such a case is often encountered in clinical practice. Oligohydramnios is often to describe pregnancies with AFI <5 and borderline/ low normal amniotic fluid volume to describe pregnancies with AFI 5 to 8 (4) Alternatively, some clinicians prefer the single vertical pocket (SVP) with severe oligohydramnios defined as SVP less than 1 and mild oligohydramnios defined as SVP 1 to 2 Aim of this current study sought to determine the relation of fetal renal artery Doppler with normal pregnancies and pregnancies complicated by oligohydramnios.Methods: Sample size was 50 patients in the 2nd trimester; they subdivided into two groups based on the amount of the amniotic fluid as follow: Group I: Control group (AFI 5-25) N = 25 cases.Group II: Oligohydramnios group (AFI less than 5) N = 25 cases.All patients in this study subjected to the following:Ultrasound study: Measurement of fetal biometry, Detection of congenital malformation. And Amniotic fluid index measurement. Doppler study: Renal artery (RA) and Umbilical artery (UA).Results: There was statistically significant difference between groups according to Renal and umbilical artery Doppler indices PI & RI in Gestational Age 26 weeks, statistically significant difference over the periods through renal artery Doppler indices RI, statistically significant difference over the periods through umbilical artery Doppler indices PI in each group and Renal Artery PI Which was 2.681, with sensitivity of 44.6% specificity of 53% with diagnostic accuracy of 47%, (p-value >0.05 NS). Renal Artery RI Which was 0.989, with sensitivity of 43.5% specificity of 43% with diagnostic accuracy of 33%, (p-value >0.05 NS). Umbilical Artery PI Which was 1.044, with sensitivity of 42.1% specificity of 39.5% with diagnostic accuracy of 29.7%, (p-value >0.05 NS). Umbilical Artery RI Which was 0.645, with sensitivity of 48.5% specificity of 42.1% with diagnostic accuracy of 41.6%, (p-value >0.05 NS). Conclusions: We recommend introducing renal artery resistance index as auxiliary ante partum test for women with Amniotic fluid abnormalities in addition to other ante partum fetal wellbeing tests to predict fetuses at risk of hypoxia.

Pharmacology of Mammalian Na+-Dependent Transporters of Inorganic Phosphate.

Inorganic phosphate (Pi) is an essential component of many biologically important molecules such as DNA, RNA, ATP, phospholipids, or apatite. It is required for intracellular phosphorylation signaling events and acts as pH buffer in intra- and extracellular compartments. Intestinal absorption, uptake into cells, and renal reabsorption depend on a set of different phosphate transporters from the SLC20 (PiT transporters) and SLC34 (NaPi transporters) gene families. The physiological relevance of these transporters is evident from rare monogenic disorders in humans affecting SLC20A2 (Fahr's disease, basal ganglia calcification), SLC34A1 (idiopathic infantile hypercalcemia), SLC34A2 (pulmonary alveolar microlithiasis), and SLC34A3 (hereditary hypophosphatemic rickets with hypercalciuria). SLC34 transporters are inhibited by millimolar concentrations of phosphonoformic acid or arsenate while SLC20 are relatively resistant to these compounds. More recently, a series of more specific and potent drugs have been developed to target SLC34A2 to reduce intestinal Pi absorption and to inhibit SLC34A1 and/or SLC34A3 to increase renal Pi excretion in patients with renal disease and incipient hyperphosphatemia. Also, SLC20 inhibitors have been developed with the same intention. Some of these substances are currently undergoing preclinical and clinical testing. Tenapanor, a non-absorbable Na+/H+-exchanger isoform 3 inhibitor, reduces intestinal Pi absorption likely by indirectly acting on the paracellular pathway for Pi and has been tested in several phase III trials for reducing Pi overload in patients with renal insufficiency and dialysis.

Does the composition of urinary extracellular vesicles reflect the abundance of renal Na+/phosphate transporters?

Studies addressing homeostasis of inorganic phosphate (Pi) are mostly restricted to murine models. Data provided by genetically modified mice suggest that renal Pi reabsorption is primarily mediated by the Na+/Pi cotransporter NaPi-IIa/Slc34a1, whereas the contribution of NaPi-IIc/Slc34a3 in adult animals seems negligible. However, mutations in both cotransporters associate with hypophosphatemic syndromes in humans, suggesting major inter-species heterogeneity. Urinary extracellular vesicles (UEV) have been proposed as an alternative source to analyse the intrinsic expression of renal proteins in vivo. Here, we analyse in rats whether the protein abundance of renal Pi transporters in UEV correlates with their renal content. For that, we compared the abundance of NaPi-IIa and NaPi-IIc in paired samples from kidneys and UEV from rats fed acutely and chronically on diets with low or high Pi. In renal brush border membranes (BBM) NaPi-IIa was detected as two fragments corresponding to the full-length protein and to a proteolytic product, whereas NaPi-IIc migrated as a single full-length band. The expression of NaPi-IIa (both fragments) in BBM adapted to acute as well to chronic changes of dietary Pi, whereas adaptation of NaPi-IIc was only detected in response to chronic administration. Both transporters were detected in UEV as well. UEV reflected the renal adaptation of the NaPi-IIa proteolytic fragment (but not the full-length protein) upon chronic but not acute dietary changes, while also reproducing the chronic regulation of NaPi-IIc. Thus, the composition of UEV reflects only partially changes in the expression of NaPi-IIa and NaPi-IIc at the BBM triggered by dietary Pi.

Acute adaptation of renal phosphate transporters in the murine kidney to oral phosphate intake requires multiple signals.

Dietary inorganic phosphate (Pi) modulates renal Pi reabsorption by regulating the expression of the NaPi-IIa and NaPi-IIc Pi transporters. Here, we aimed to clarify the role of several Pi-regulatory mechanisms including parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23) and inositol hexakisphosphate kinases (IP6-kinases) in the acute regulation of NaPi-IIa and NaPi-IIc. Wildtype (WT) and PTH-deficient mice (PTH-KO) with/without inhibition of FGF23 signalling were gavaged with Pi/saline and examined at 1, 4 and 12h. Pi-gavage elevated plasma Pi and decreased plasma Ca2+ in both genotypes after 1h Within 1h, Pi-gavage decreased NaPi-IIa abundance in WT and PTH-KO mice. NaPi-IIc was downregulated 1h post-administration in WT and after 4h in PTH-KO. PTH increased after 1h in WT animals. After 4h Pi-gavage, FGF23 increased in both genotypes being higher in the KO group. PTHrp and dopamine were not altered by Pi-gavage. Blocking FGF23 signalling blunted PTH upregulation in WT mice and reduced NaPi-IIa downregulation in PTH-KO mice 4h after Pi-gavage. Inhibition of IP6-kinases had no effect. (1) Acute downregulation of renal Pi transporters in response to Pi intake occurs also in the absence of PTH and FGF23 signalling, (2) when FGF23 signalling is blocked, a partial contribution of PTH is revealed, (3) IP6 kinases, intracellular Pi-sensors in yeast and bacteria, are not involved, and (4) Acute Pi does not alter PTHrp and dopamine. Thus, signals other than PTH, PTHrp, FGF23 and dopamine contribute to renal adaption.

RGS14 regulates PTH- and FGF23-sensitive NPT2A-mediated renal phosphate uptake via binding to the NHERF1 scaffolding protein

Phosphate homeostasis, mediated by dietary intake, renal absorption, and bone deposition, is incompletely understood because of the uncharacterized roles of numerous implicated protein factors. Here, we identified a novel role for one such element, regulator of G protein signaling 14 (RGS14), suggested by genome-wide association studies to associate with dysregulated Pi levels. We show that human RGS14 possesses a carboxy-terminal PDZ ligand required for sodium phosphate cotransporter 2a (NPT2A) and sodium hydrogen exchanger regulatory factor-1 (NHERF1)–mediated renal Pi transport. In addition, we found using isotope uptake measurements combined with bioluminescence resonance energy transfer assays, siRNA knockdown, pull-down and overlay assays, and molecular modeling that secreted proteins parathyroid hormone (PTH) and fibroblast growth factor 23 inhibited Pi uptake by inducing dissociation of the NPT2A–NHERF1 complex. PTH failed to affect Pi transport in cells expressing RGS14, suggesting that it suppresses hormone-sensitive but not basal Pi uptake. Interestingly, RGS14 did not affect PTH-directed G protein activation or cAMP formation, implying a postreceptor site of action. Further pull-down experiments and direct binding assays indicated that NPT2A and RGS14 bind distinct PDZ domains on NHERF1. We showed that RGS14 expression in human renal proximal tubule epithelial cells blocked the effects of PTH and fibroblast growth factor 23 and stabilized the NPT2A–NHERF1 complex. In contrast, RGS14 genetic variants bearing mutations in the PDZ ligand disrupted RGS14 binding to NHERF1 and subsequent PTH-sensitive Pi transport. In conclusion, these findings identify RGS14 as a novel regulator of hormone-sensitive Pi transport. The results suggest that changes in RGS14 function or abundance may contribute to the hormone resistance and hyperphosphatemia observed in kidney diseases.

MO566THE BONE EXPRESSION OF WNT INHIBITORS IN THE EXPERIMENTAL MODEL OF EARLY CHRONIC KIDNEY DISEASE

Abstract Background and Aims Molecular mechanisms implicated in the initial stages of inorganic phosphate (Pi) imbalance in chronic kidney disease (CKD) remain poorly understood.The aim of the study was to evaluate whether canonical Wnt pathway inhibitors (iWnt) involved in early response to Pi retention in CKD. Methods Mild CKD was induced by 3/4 nephrectomy (NE) in spontaneously hypertensive rats (SHR) fed rat chow diet containing 0.6 % phosphate. Controls were sham operated SHR (SO). Duration of experimental exposure (NE or SO) was 2 and 6 months. Serum levels of creatinine (Cr), inorganic phosphate (Pi), fractional Pi excretion (FEPi), intact parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), alfa-Klotho (KL), sclerostin (SOST) and Dickkopf-1 (DKK1) were measured. The following morphological characteristics by light microscopy of bone metaphysis and kidney tissues: the area of renal interstitial fibrosis (RF) (Masson's trichrome), bone matrix volume (MV), the active osteoblasts to trabecular cells number ratio (aOB/cells), eroded surface to bone surface ratio (ES/BS) (hematoxylin &amp; eosin), and bone SOST and DKK1 proteins expression (by IHC) were analyzed and calculated quantitatively. Statistical comparisons among groups were performed using Mann–Whitney U-test and Kruskal-Wallis H-test. Results Serum Cr, RF and indices of Pi exchange in the experimental model corresponded to early CKD (Table). Pi elevated in NE6 suggestive for its renal retention. KL level decreased (Table) in all experimental groups vs control. No differences were observed in serum levels FGF23 (p=0.62) and PTH (p=0.63). Serum SOST and DKK1 levels were significantly higher in NE6 group compared to SO6 (Table). The bone SOST and DKK1 expression increased in NE6 compared to SO6 (Figure). aOB/cells were lower in NE2, SO6 and NE6 vs SO2 (all p-values&amp;lt;=0.041). ES/BS increased in NE2 (vs SO2) while being lowest in NE6 and SO6 animals (Table). SOST and DKK1 metaphyseal expression increased in NE6 compared to SO2, SO6, NE2 (Figure). Osteocyte SOST expression increased in SO6 compared to SO2 and NE2 without differences in later groups. Osteoblast SOST expression was also higher in SO6 vs SO2 (Figure). Conclusion Increased serum levels of sclerostin and Dickkopf-1 and their bone expression are apparent in early stages of experimental CKD associating with hyperphosphatemia. Alterations of bone resorption and osteoblast depopulation occurred before the increase of serum Pi likely reflecting incipient stages of renal Pi retention. These molecular and cellular events seem to be independent of systemic FGF23 and PTH response.

Inositol hexakisphosphate kinases 1 and 2 (IP6K1/2) are involved in renal control of phosphate homeostasis

Phosphate (Pi) is an indispensable component of living organisms. In the body, Pi homeostasis is tightly regulated by four main tissues: intestine, kidney, bone and parathyroid glands. There are multiple complex mechanisms underlying the regulation of serum Pi including hormones and transporters. Although plenty of studies have analysed Pi regulation, the mechanism with which Pi levels are sensed by mammalian cells remains elusive. Molecular mechanisms of Pi-sensing have been elucidated in detail in yeast and bacteria. However, most of the Pi-sensing proteins identified in yeast and bacteria are encoded by genes that are not conserved in mammals. As a major exception, inositol polyphosphates—which respond to changes in ambient Pi in yeast and, in turn, regulate the expression of Pi transporters—are also found in mammals. Here, we addressed the role of inositol polyphosphates and their biogenic enzymes, the IP6Ks (especially IP6K1 and 2), in cellular Pi sensing in mammals. Human and mouse kidneys express the IP6K1 and IP6K2 isoforms. Likewise, Opossum Kidney (OK) cells, a model for renal proximal tubules express IP6K1 and 2. Pharmacological inhibition of IP6K1/2 or CRISPR/Cas9 mediated knockout in OK cells downregulates Pi transporter mRNA, particularly NaPi-IIa/c. Ip6k1-/-2-/- KO cells also exhibited decreased Pi uptake (by 70%) and an abrogated adaptation to ambient Pi. To study the role of IP6K1 and 2 in vivo, we generated renal tubule-specific inducible ip6k1-/-2-/- KO mice: the mice had strongly reduced expression of renal Pi transporters, at the protein and mRNA levels, and lower serum Pi (by 20%) together with the transcriptional downregulation of many other transporters. Serum intact fibroblast growth factor 23—a principal phosphaturic hormone—was also substantially reduced (10-fold) in these mice. Our data indicate that the deletion of IP6K1 and 2 decreases transcription of renal Pi transporters both in vivo and in vitro, and that Ip6k1-/-2-/- cells are unable to adapt to ambient Pi. Therefore, IP6K1 and 2 play a critical role in renal Pi metabolism and, potentially, sensing.

Characterization of Sodium‐Dependent Pi Transport Inhibition by NAD

Hyperphosphatemia during end-stage renal disease causes many adverse outcomes. To prevent them, several Pi intestinal absorption inhibitors are necessary for targeting either the paracellular (diffusion) or the transcellular (transport) absorption pathway. Nicotinamide adenine dinucleotide (NADH/NAD+) is a classical inhibitor of intestinal and renal Pi transport and is a circadian modulator of phosphatemia. The mechanism of NAD transporter inhibition remains unclear, although a competitive inhibition mechanism has been suggested. In this work, we sought to determine if this mechanism has a direct effect on Pi transporters. The five known rat Na/Pi cotransporters (NaPi2a, NaPi2b, NaPi2c, PiT1, and PiT2) were expressed in Xenopus laevis oocytes. Uptake of 32P-Pi was performed for three days post-injection of 5 ng of the corresponding cRNAs per oocyte and after 30 minutes of preincubation with 0.5 mM NAD, NADH, or NAM. None of the compounds affected the Pi uptake of any of the expressed Pi transporters in oocytes. Pi transport in the brush border membrane vesicles (BBMV) of either rat kidney cortex or jejunum epithelium was also assayed. As expected, it was significantly inhibited by NADH and NAD when BBMV were preincubated for 30 min at room or ice-cold temperature. NAM had no effect, and the effect was specific for Na-dependent Pi uptake (D-glucose uptake was not affected). Dose-response analyses on jejunum or kidney BBMV revealed IC50 values in the micromolar range for 50 µM of Pi uptake. Several Michaelis-Menten kinetics were performed in the presence of different concentrations of NAD, and a Lineweaver-Burk plot suggested changes in affinity (i.e., competitive inhibition), which contrasts with the lack of an effect on the transporters expressed in oocytes. The competitive model was confirmed by non-linear regression of a global fit with shared parameters, providing a Ki value of 538 µM NAD. When the effect of NAD was assayed in Opossum Kidney cells, a proximal tubule cell line model expressing a regulated NaPi2a, no evidence of Pi transport inhibition was observed. To check whether NAD-mediated ribosylation was involved in Pi transport inhibition, a classical ribosylation assay was performed with 32P-NAD, using jejunum and renal BBMV. No evidence of ribosylation was found for NaPi2b or NaPi2a, even after one month of exposure to an X-ray film. This was confirmed using several ribosylation inhibitors (meta-iodobenzylguanidine, novobiocin, vitamin K1, vitamin K3, or 3-methoxybenzamide), which failed to prevent NAD inhibition of Pi transport in BBMV. In conclusion, despite the kinetic findings in BBMV, NAD inhibition of Na/Pi-cotransporters is, most likely, not mediated through a direct interaction with these transporters. Instead, inhibition seems to be mediated through an indirect mechanism acting on components that are absent in Xenopus oocytes and in OK cells, which ends in the modification of Pi transport affinity (Km) and occurs at ice-cold temperature. Nevertheless, the unlikely inhibition of a novel, unknown Pi transporter cannot be completely discarded.

Intestinal and Renal Pi Transport Adaptation and Hormonal Changes in Response to Acute Changes in Dietary Pi

A hallmark of the successful control of Pi homeostasis is the adaptation of Pi intestinal absorption and renal excretion to changes in the dietary content of Pi. Acute and chronic adaptation mechanisms have been described for both tissues, yet despite these efforts, many questions related to hormonal changes and transport systems are still unclear. Male Wistar rats were fed well-balanced fodder containing either 0.1, 0.6, or 1.2% Pi, prepared with NaH2PO4 and K2HPO4 to pH 7.4 and compensated with NaCl and KCl. Some rats were fed ad libitum for 6 days, while other animals were fed only 4 hours in the morning for 6 days. Some of the rats from this 4-hour feeding scheme that had been fed either 0.1 or 1.2% Pi fodder for 5 days received the opposite fodder on the 6th day. Food consumption, Pi ingestion, and growth were determined. Pi deficiency-related acidosis was minimally observed in the animals that, after 5 days eating 1.2% Pi food, were switched to 0.1% Pi on the 6thday: blood pH was reduced to 7.32, bicarbonate fell to 25.2 mM, and the base excess dropped to -1.74 mM. Pi transport in jejunum and kidney cortex brush border membrane vesicles (BBMV) was always higher in the animals chronically fed with 0.1% Pi, and it was similar at 0.6 and 1.2% Pi. In the case of animals whose diet was switched the last day, there were no differences in jejunum BBMV Pi transport, in contrast to previous studies that have shown a higher transport rate after switching to a high Pi content. In kidney BBMV, Pi transport was higher in the change from 1.2 to 0.1% Pi. Plasma Pi was always proportional to the fodder Pi content. Plasma FGF23 and PTH concentrations varied similarly and were always proportional to the ingested Pi in all groups, including the animals whose food was switched, meaning that both hormones changed after 4 hours of high Pi intake. Differences in the phosphatonins sFR4 and MEPE were not significant. Also, no differences were observed in dopamine, corticosterone, insulin, thyroxine, calcitriol, or Klotho. A similar experiment was performed in parathyroidectomized (PTX) rats, but only using the 4-hour feeding regime. Food consumption and growth was similar in sham and PTX animals. Blood plasma Pi was higher in PTX animals in all groups except for those chronically fed at 0.1% Pi. Transport in jejunum BBMV was similar in sham and PTX rats, and NaPi2b protein abundance correlated with the transport rate. In kidney BBMV, all groups showed higher transport rates in PTX rats (except for the chronic 0.1% Pi group, in which the rates were similar), and no differences were observed between the animals whose food was acutely switched the last day, therefore confirming the role of PTH over the relevance of FGF23 in the kidney's acute response. All hormones behaved similarly in sham and PTX animals, except for dopamine, which was very low in the 0.1%-Pi fed PTX rats, and FGF23 which was surprisingly lower in all groups compared to sham rats. In conclusion, a pH- and ion-balanced fodder prevented the paradoxical increase of Pi intestinal uptake when a Pi-enriched food was given. This food also prevented the strong metabolic acidosis associated with experimentally Pi-depleted fodder. Acute adaptation only occurs in the kidney, and PTH is the hormone that mediates it.

Expression of NaPi-IIb in rodent and human kidney and upregulation in a model of chronic kidney disease.

Na+-coupled phosphate cotransporters from the SLC34 and SLC20 families of solute carriers mediate transepithelial transport of inorganic phosphate (Pi). NaPi-IIa/Slc34a1, NaPi-IIc/Slc34a3, and Pit-2/Slc20a2 are all expressed at the apical membrane of renal proximal tubules and therefore contribute to renal Pi reabsorption. Unlike NaPi-IIa and NaPi-IIc, which are rather kidney-specific, NaPi-IIb/Slc34a2 is expressed in several epithelial tissues, including the intestine, lung, testis, and mammary glands. Recently, the expression of NaPi-IIb was also reported in kidneys from rats fed on high Pi. Here, we systematically quantified the mRNA expression of SLC34 and SLC20 cotransporters in kidneys from mice, rats, and humans. In all three species, NaPi-IIa mRNA was by far the most abundant renal transcript. Low and comparable mRNA levels of the other four transporters, including NaPi-IIb, were detected in kidneys from rodents and humans. In mice, the renal expression of NaPi-IIa transcripts was restricted to the cortex, whereas NaPi-IIb mRNA was observed in medullary segments. Consistently, NaPi-IIb protein colocalized with uromodulin at the luminal membrane of thick ascending limbs of the loop of Henle segments. The abundance of NaPi-IIb transcripts in kidneys from mice was neither affected by dietary Pi, the absence of renal NaPi-IIc, nor the depletion of intestinal NaPi-IIb. In contrast, it was highly upregulated in a model of oxalate-induced kidney disease where all other SLC34 phosphate transporters were downregulated. Thus, NaPi-IIb may contribute to renal phosphate reabsorption, and its upregulation in kidney disease might promote hyperphosphatemia.

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.

Skeletal mineralization: mechanisms and diseases.

Skeletal mineralization is initiated in matrix vesicles (MVs), the small extracellular vesicles derived from osteoblasts and chondrocytes. Calcium and inorganic phosphate (Pi) taken up by MVs form hydroxyapatite crystals, which propagate on collagen fibrils to mineralize the extracellular matrix. Insufficient calcium or phosphate impairs skeletal mineralization. Because active vitamin D is necessary for intestinal calcium absorption, vitamin D deficiency is a significant cause of rickets/osteomalacia. Chronic hypophosphatemia also results in rickets/osteomalacia. Excessive action of fibroblast growth factor 23 (FGF23), a key regulator of Pi metabolism, leads to renal Pi wasting and impairs vitamin D activation. X-linked hypophosphatemic rickets (XLH) is the most common form of hereditary FGF23-related hypophosphatemia, and enhanced FGF receptor (FGFR) signaling in osteocytes may be involved in the pathogenesis of this disease. Increased extracellular Pi triggers signal transduction via FGFR to regulate gene expression, implying a close relationship between Pi metabolism and FGFR. An anti-FGF23 antibody, burosumab, has recently been developed as a new treatment for XLH. In addition to various forms of rickets/osteomalacia, hypophosphatasia (HPP) is characterized by impaired skeletal mineralization. HPP is caused by inactivating mutations in tissue-nonspecific alkaline phosphatase, an enzyme rich in MVs. The recent development of enzyme replacement therapy using bone-targeting recombinant alkaline phosphatase has improved the prognosis, motor function, and quality of life in patients with HPP. This links impaired skeletal mineralization with various conditions, and unraveling its pathogenesis will lead to more precise diagnoses and effective treatments.

Inhibitors of canonical Wnt signaling pathway and inorganic phosphate imbalance in experimental chronic kidney disease

BACKGROUND. The molecular mechanisms of the initial stages of inorganic phosphate (Pi) metabolic disorders in chronic kidney disease (CKD) remain poorly understood.THE AIM. To test the hypothesis about changes in canonical Wnt signaling pathway inhibitors biosynthesis and a concomitant decrease in bone turnover as one of early mechanisms of Pi imbalance in CKD.MATERIAL AND METHODS. Creatinine (Cr), inorganic phosphate (Pi), serum parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), osteoprotegerin (OPG), sclerostin (SOST) and Dickkopf-1 (DKK), renal SOST and DKK mRNA expression, albuminuria (Alb), proteinuria (uTP) levels, fractional (FEPi) and daily (uPi24) Pi excretion were analyzed in SHR rats (N = 52) with 3/4 nephrectomy (NE) or sham operation (SO) and observation periods of 2, 4, and 6 months.RESULTS. Experimental model was comparable with 1-2 stages of CKD. In groups NE4 and NE6, the concentration of sPi and renal Pi excretion (FEPi and uPi24) were significantly higher vs corresponding controls SO4 (p = 0.006, p &lt;0.010) and SO6 (p = 0.002, p = 0.028). Serum concentrations of FGF23 and PTH in NE and SO animals did not change significantly. In NE4 and NE6 groups, serum SOST and DKK concentrations were significantly higher vs controls (p &lt;0.049, p &lt;0.043), while the kidney expression SOST and DKK mRNA in NE rats did not change significantly or decreased (p = 0.002, p &lt;0.011). The serum concentration of OPG was higher in the NE6 vs SO6 control (p = 0.028).CONCLUSION. The initial stages of experimental CKD are characterized by an increase in serum concentrations of Dikkopf-1, sclerostin and osteoprotegerin. The obtained data suggest the possible role of canonical Wnt signaling inhibition and reduction of bone turnover in the pathogenesis of Pi metabolic disorders in early stages of CKD.