Phosphate Restriction Prevents Metabolic Acidosis and Curbs Rise in FGF23 and Mortality in Murine Folic Acid-Induced AKI.

SIGNIFICANCE STATEMENT: Patients with AKI suffer a staggering mortality rate of approximately 30%. Fibroblast growth factor 23 (FGF23) and phosphate (P i ) rise rapidly after the onset of AKI and have both been independently associated with ensuing morbidity and mortality. This study demonstrates that dietary P i restriction markedly diminished the early rise in plasma FGF23 and prevented the rise in plasma P i , parathyroid hormone, and calcitriol in mice with folic acid-induced AKI (FA-AKI). Furthermore, the study provides evidence for P i -sensitive osseous Fgf23 mRNA expression and reveals that P i restriction mitigated calciprotein particles (CPPs) formation, inflammation, acidosis, cardiac electrical disturbances, and mortality in mice with FA-AKI. These findings suggest that P i restriction may have a prophylactic potential in patients at risk for AKI. BACKGROUND: In AKI, plasma FGF23 and P i rise rapidly and are independently associated with disease severity and outcome. METHODS: The effects of normal (NP) and low (LP) dietary P i were investigated in mice with FA-AKI after 3, 24, and 48 hours and 14 days. RESULTS: After 24 hours of AKI, the LP diet curbed the rise in plasma FGF23 and prevented that of parathyroid hormone and calcitriol as well as of osseous but not splenic or thymic Fgf23 mRNA expression. The absence of Pth prevented the rise in calcitriol and reduced the elevation of FGF23 in FA-AKI with the NP diet. Furthermore, the LP diet attenuated the rise in renal and plasma IL-6 and mitigated the decline in renal α -Klotho. After 48 hours, the LP diet further dampened renal IL-6 expression and resulted in lower urinary neutrophil gelatinase-associated lipocalin. In addition, the LP diet prevented the increased formation of CPPs. Fourteen days after AKI induction, the LP diet group maintained less elevated plasma FGF23 levels and had greater survival than the NP diet group. This was associated with prevention of metabolic acidosis, hypocalcemia, hyperkalemia, and cardiac electrical disturbances. CONCLUSIONS: This study reveals P i -sensitive FGF23 expression in the bone but not in the thymus or spleen in FA-AKI and demonstrates that P i restriction mitigates CPP formation, inflammation, acidosis, and mortality in this model. These results suggest that dietary P i restriction could have prophylactic potential in patients at risk for AKI.

The Ip6k1 and Ip6k2 Kinases Are Critical for Normal Renal Tubular Function.

SIGNIFICANCE STATEMENT: Kidneys are gatekeepers of systemic inorganic phosphate balance because they control urinary phosphate excretion. In yeast and plants, inositol hexakisphosphate kinases (IP6Ks) are central to regulate phosphate metabolism, whereas their role in mammalian phosphate homeostasis is mostly unknown. We demonstrate in a renal cell line and in mice that Ip6k1 and Ip6k2 are critical for normal expression and function of the major renal Na + /Pi transporters NaPi-IIa and NaPi-IIc. Moreover, Ip6k1/2-/- mice also show symptoms of more generalized kidney dysfunction. Thus, our results suggest that IP6Ks are essential for phosphate metabolism and proper kidney function in mammals. BACKGROUND: Inorganic phosphate is an essential mineral, and its plasma levels are tightly regulated. In mammals, kidneys are critical for maintaining phosphate homeostasis through mechanisms that ultimately regulate the expression of the Na + /Pi cotransporters NaPi-IIa and NaPi-IIc in proximal tubules. Inositol pyrophosphate 5-IP 7 , generated by IP6Ks, is a main regulator of phosphate metabolism in yeast and plants. IP6Ks are conserved in mammals, but their role in phosphate metabolism in vivo remains unexplored. METHODS: We used in vitro (opossum kidney cells) and in vivo (renal tubular-specific Ip6k1/2-/- mice) models to analyze the role of IP6K1/2 in phosphate homeostasis in mammals. RESULTS: In both systems, Ip6k1 and Ip6k2 are responsible for synthesis of 5-IP 7 . Depletion of Ip6k1/2 in vitro reduced phosphate transport and mRNA expression of Na + /Pi cotransporters, and it blunts phosphate transport adaptation to changes in ambient phosphate. Renal ablation of both kinases in mice also downregulates the expression of NaPi-IIa and NaPi-IIc and lowered the uptake of phosphate into proximal renal brush border membranes. In addition, the absence of Ip6k1 and Ip6k2 reduced the plasma concentration of fibroblast growth factor 23 and increased bone resorption, despite of which homozygous males develop hypophosphatemia. Ip6k1/2-/- mice also show increased diuresis, albuminuria, and hypercalciuria, although the morphology of glomeruli and proximal brush border membrane seemed unaffected. CONCLUSIONS: Depletion of renal Ip6k1/2 in mice not only altered phosphate homeostasis but also dysregulated other kidney functions.

Absence of claudin-3 does not alter intestinal absorption of phosphate in mice.

Intestinal absorption of phosphate is bimodal, consisting of a transcellular pathway and a poorly characterized paracellular mode, even though the latter one contributes to the bulk of absorption under normal dietary conditions. Claudin-3 (Cldn3), a tight junction protein present along the whole intestine in mice, has been proposed to tighten the paracellular pathway for phosphate. The aim of this work was to characterize the phosphate-related phenotype of Cldn3-deficient mice. Cldn3-deficient mice and wildtype littermates were fed standard diet or challenged for 3 days with high dietary phosphate. Feces, urine, blood, intestinal segments and kidneys were collected. Measurements included fecal, urinary, and plasma concentrations of phosphate and calcium, plasma levels of phosphate-regulating hormones, evaluation of trans- and paracellular phosphate transport across jejunum and ileum, and analysis of intestinal phosphate and calcium permeabilities. Fecal and urinary excretion of phosphate as well as its plasma concentration was similar in both genotypes, under standard and high-phosphate diet. However, Cldn3-deficient mice challenged with high dietary phosphate had a reduced urinary calcium excretion and increased plasma levels of calcitriol. Intact FGF23 concentration was also similar in both groups, regardless of the dietary conditions. We found no differences either in intestinal phosphate transport (trans- or paracellular) and phosphate and calcium permeabilities between genotypes. The intestinal expression of claudin-7 remained unaltered in Cldn3-deficient mice. Our data do not provide evidence for a decisive role of Cldn3 for intestinal phosphate absorption and phosphate homeostasis. In addition, our data suggest a novel role of Cldn3 in regulating calcitriol levels.

1,25(OH)2 vitamin D3 stimulates active phosphate transport but not paracellular phosphate absorption in mouse intestine.

KEY POINTS: Intestinal absorption of phosphate proceeds via an active/transcellular route mostly mediated by NaPi-IIb/Slc34a2 and a poorly characterized passive/paracellular pathway. Intestinal phosphate absorption and expression of NaPi-IIb are stimulated by 1,25(OH)2 vitamin D3 but whether NaPi-IIb is the only target under hormonal control remains unknown. We report that administration of 1,25(OH)2 vitamin D3 to wild-type mice resulted in the expected increase in active transport of phosphate in jejunum, without changing paracellular fluxes. Instead, the same treatment failed to alter phosphate transport in intestinal-depleted Slc34a2-deficient mice. In both genotypes, 1,25(OH)2 vitamin D3 induced similar hyperphosphaturic responses and changes in the plasma levels of FGF23 and PTH. While urinary phosphate loss induced by administration of 1,25(OH)2 vitamin D3 did not alter plasma phosphate, further studies should investigate whether chronic administration would lead to phosphate imbalance in mice with reduced active intestinal absorption. ABSTRACT: Intestinal absorption of phosphate is stimulated by 1,25(OH)2 vitamin D3. At least two distinct mechanisms underlie phosphate absorption in the gut, an active transcellular transport requiring the Na+ /phosphate cotransporter NaPi-IIb/Slc34a2, and a poorly characterized paracellular passive pathway. 1,25(OH)2 vitamin D3 stimulates NaPi-IIb expression and function, and loss of NaPi-IIb reduces intestinal phosphate absorption. However, it is remains unknown whether NaPi-IIb is the only target for hormonal regulation by 1,25(OH)2 vitamin D3 . Here we compared the effects of intraperitoneal administration of 1,25(OH)2 vitamin D3 (2 days, once per day) in wild-type and intestinal-specific Slc34a2-deficient mice, and analysed trans- vs. paracellular routes of phosphate absorption. We found that treatment stimulated active transport of phosphate only in jejunum of wild-type mice, though NaPi-IIb protein expression was upregulated in jejunum and ileum. In contrast, 1,25(OH)2 vitamin D3 administration had no effect in Slc34a2-deficient mice, suggesting that the hormone specifically regulates NaPi-IIb expression. In both groups, 1,25(OH)2 vitamin D3 elicited the expected increase of plasma fibroblast growth factor 23 (FGF23) and reduction of parathyroid hormone (PTH). Treatment resulted in hyperphosphaturia (and hypercalciuria) in both genotypes, though mice remained normophosphataemic. While increased intestinal absorption and higher FGF23 can trigger the hyperphosphaturic response in wild types, only higher FGF23 can explain the renal response in Slc34a2-deficient mice. Thus, 1,25(OH)2 vitamin D3 stimulates intestinal phosphate absorption by acting on the active transcellular pathway mostly mediated by NaPi-IIb while the paracellular pathway appears not to be affected.

Fibroblast growth factor 23 leads to endolysosomal routing of the renal phosphate cotransporters NaPi-IIa and NaPi-IIc in vivo.

Na+-dependent phosphate cotransporters NaPi-IIa and NaPi-IIc, located at the brush-border membrane of renal proximal tubules, are regulated by numerous factors, including fibroblast growth factor 23 (FGF23). FGF23 downregulates NaPi-IIa and NaPi-IIc abundance after activating a signaling pathway involving phosphorylation of ERK1/2 (phospho-ERK1/2). FGF23 also downregulates expression of renal 1-α-hydroxylase (Cyp27b1) and upregulates 24-hydroxylase (Cyp24a1), thus reducing plasma calcitriol levels. Here, we examined the time course of FGF23-induced internalization of NaPi-IIa and NaPi-IIc and their intracellular pathway toward degradation in vivo. Mice were injected intraperitoneally with recombinant human (rh)FGF23 in the absence (biochemical analysis) or presence (immunohistochemistry) of leupeptin, an inhibitor of lysosomal proteases. Phosphorylation of ERK1/2 was enhanced 60 min after rhFGF23 administration, and increased phosphorylation was still detected 480 min after injection. Colocalization of phospho-ERK1/2 with NaPi-IIa was seen at 60 and 120 min and partly at 480 min. The abundance of both cotransporters was reduced 240 min after rhFGF23 administration, with a further reduction at 480 min. NaPi-IIa and NaPi-IIc were found to colocalize with clathrin and early endosomal antigen 1 as early as 120 min after rhFGF23 injection. Both cotransporters partially colocalized with cathepsin B and lysosomal-associated membrane protein-1, markers of lysosomes, 120 min after rhFGF23 injection. Thus, NaPi-IIa and NaPi-IIc are internalized within 2 h upon rhFGF23 injection. Both cotransporters share the pathway of clathrin-mediated endocytosis that leads first to early endosomes, finally resulting in trafficking toward the lysosome as early as 120 min after rhFGF23 administration.NEW & NOTEWORTHY The hormone fibroblast growth factor 23 (FGF23) controls phosphate homeostasis by regulating renal phosphate excretion. FGF23 acts on several phosphate transporters in the kidney. Here, we define the time course of this action and demonstrate how phosphate transporters NaPi-IIa and NaPi-IIc are internalized.

Erythropoietin stimulates fibroblast growth factor 23 (FGF23) in mice and men.

Fibroblast growth factor 23 (FGF23) is a major endocrine regulator of phosphate and 1,25 (OH)2 vitamin D3 metabolism and is mainly produced by osteocytes. Its production is upregulated by a variety of factors including 1,25 (OH)2 vitamin D3, high dietary phosphate intake, and parathyroid hormone (PTH). Recently, iron deficiency and hypoxia have been suggested as additional regulators of FGF23 and a role of erythropoietin (EPO) was shown. However, the regulation of FGF23 by EPO and the impact on phosphate and 1,25(OH)2 vitamin D3 are not completely understood. Here, we demonstrate that acute administration of recombinant human EPO (rhEPO) to healthy humans increases the C-terminal fragment of FGF23 (C-terminal FGF23) but not intact FGF23 (iFGF23). In mice, rhEPO stimulates acutely (24 h) C-terminal FGF23 but iFGF23 only after 4 days without effects on PTH and plasma phosphate. 1,25 (OH)2 D3 levels and αklotho expression in the kidney decrease after 4 days. rhEPO induced FGF23 mRNA in bone marrow but not in bone, with increased staining of FGF23 in CD71+ erythroid precursors in bone marrow. Chronic elevation of EPO in transgenic mice increases iFGF23. Finally, acute injections of recombinant FGF23 reduced renal EPO mRNA expression. Our data demonstrate stimulation of FGF23 levels in mice which impacts mostly on 1,25 (OH)2 vitamin D3 levels and metabolism. In humans, EPO is mostly associated with the C-terminal fragment of FGF23; in mice, EPO has a time-dependent effect on both FGF23 forms. EPO and FGF23 may form a feedback loop controlling and linking erythropoiesis and mineral metabolism.

The murine Cl⁻/HCO⁻3 exchanger Ae3 (Slc4a3) is not required for acid-base balance but is involved in magnesium handling by the kidney.

BACKGROUND: The Slc4 family of bicarbonate transporters consists of several members, many of which are highly expressed in the kidney and play an important role in acid-base homeostasis. Among them are Ae1 (Slc4a1) and Ae2 (Slc4a2). Another member, Ae3 (Slc4a3), is suggested to be expressed in the kidney, however, its localization and impact on renal function is still unknown. Ae3 has also been implicated in the central control of breathing. AIMS: Here, we analyzed the expression of Ae3 transcripts in isolated nephron segments and investigated systemic and renal acid-base homeostasis and renal electrolyte handling in the absence of Ae3, using a knock out mouse model. METHODS: qPCR was used to localize Ae3 transcripts in the murine nephron, metabolic studies and whole body plethysmography to assess the role of Ae3 in renal functions. RESULTS: Two Ae3 transcripts, the brain variant bAe3 and the cardiac variant cAe3, are expressed at low levels in the murine kidney. Although differentially distributed, they localize mostly to the distal nephron and renal collecting duct system. At baseline and after an acid challenge, mice deficient for Ae3 did not show major disturbances in renal acid-base excretion. Respiratory responses in whole body plethysmography to acid loading and CO2 and O2 challenges were normal. No major differences in renal electrolyte handling were discovered except for small changes in magnesium, potassium and sodium excretion after 7 days of acid loading. We therefore challenged mice with diets with high and low magnesium diets and found no differences in renal magnesium excretion but elevated expression of the Trpm6 magnesium channel in Ae3 KO mice. In conclusion, Ae3 is expressed in murine kidney at very low levels. CONCLUSIONS: Ae3 plays no role in systemic acid-base homeostasis but may modify renal magnesium handling inducing a higher expression of Trpm6.

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

AIMS: 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. METHODS: 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 12 h. RESULTS: Pi-gavage elevated plasma Pi and decreased plasma Ca2+ in both genotypes after 1 h Within 1 h, Pi-gavage decreased NaPi-IIa abundance in WT and PTH-KO mice. NaPi-IIc was downregulated 1 h post-administration in WT and after 4 h in PTH-KO. PTH increased after 1 h in WT animals. After 4 h 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 4 h after Pi-gavage. Inhibition of IP6-kinases had no effect. CONCLUSIONS: (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.

The human pathogenic 91del7 mutation in SLC34A1 has no effect in mineral homeostasis in mice.

Kidneys are key regulators of phosphate homeostasis. Biallelic mutations of the renal Na+/phosphate cotransporter SLC34A1/NaPi-IIa cause idiopathic infantile hypercalcemia, whereas monoallelic mutations were frequently noted in adults with kidney stones. Genome-wide-association studies identified SLC34A1 as a risk locus for chronic kidney disease. Pathogenic mutations in SLC34A1 are present in 4% of the general population. Here, we characterize a mouse model carrying the 91del7 in-frame deletion, a frequent mutation whose significance remains unclear. Under normal dietary conditions, 12 weeks old heterozygous and homozygous males have similar plasma and urinary levels of phosphate as their wild type (WT) littermates, and comparable concentrations of parathyroid hormone, fibroblast growth factor 23 (FGF-23) and 1,25(OH)2 vitamin D3. Renal phosphate transport, and expression of NaPi-IIa and NaPi-IIc cotransporters, was indistinguishable in the three genotypes. Challenging mice with low dietary phosphate did not result in differences between genotypes with regard to urinary and plasma phosphate. Urinary and plasma phosphate, plasma FGF-23 and expression of cotransporters were similar in all genotypes after weaning. Urinary phosphate and bone mineral density were also comparable in 300 days old WT and mutant mice. In conclusion, mice carrying the 91del7 truncation do not show signs of impaired phosphate homeostasis.

Intestinal epithelial ablation of Pit-2/Slc20a2 in mice leads to sustained elevation of vitamin D3 upon dietary restriction of phosphate.

AIM: Several Na+ -dependent phosphate cotransporters, namely NaPi-IIb/SLC34A2, Pit-1/SLC20A1 and Pit-2/SLC20A2, are expressed at the apical membrane of enterocytes but their contribution to active absorption of phosphate is unclear. The aim of this study was to compare their pattern of mRNA expression along the small and large intestine and to analyse the effect of intestinal depletion of Pit-2 on phosphate homeostasis. METHODS: Intestinal epithelial Pit-2-deficient mice were generated by crossing floxed Pit-2 with villin-Cre mice. Mice were fed 2 weeks standard or low phosphate diets. Stool, urine, plasma and intestinal and renal tissue were collected. Concentration of electrolytes and hormones, expression of mRNAs and proteins and intestinal transport of tracers were analysed. RESULTS: Intestinal mRNA expression of NaPi-IIb and Pit-1 is segment-specific, whereas the abundance of Pit-2 mRNA is more homogeneous. In ileum, NaPi-IIb mRNA expression is restricted to enterocytes, whereas Pit-2 mRNA is found in epithelial and non-epithelial cells. Overall, their mRNA expression is not regulated by dietary phosphate. The absence of Pit-2 from intestinal epithelial cells does not affect systemic phosphate homeostasis under normal dietary conditions. However, in response to dietary phosphate restriction, Pit-2-deficient mice showed exacerbated hypercalciuria and sustained elevation of 1,25(OH)2 vitamin D3 . CONCLUSIONS: In mice, the intestinal Na+ /phosphate cotransporters are not coexpressed in all segments. NaPi-IIb but not Pit-2 mRNA is restricted to epithelial cells. Intestinal epithelial Pit-2 does not contribute significantly to absorption of phosphate under normal dietary conditions. However, it may play a more significant role upon dietary phosphate restriction.