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.

Chromosome analysis of 303 pregnancy losses in Mexico.

BACKGROUND: Chromosomal abnormalities are present in 50 to 60% of miscarriages and in 6 to 19% of stillbirths. Although microarrays are preferred for studying chromosomal abnormalities, many hospitals cannot offer this methodology. OBJECTIVE: To present the results of the cytogenetic analysis of 303 products of conception (POC), which included 184 miscarriages, 49 stillbirths and 17 cases of undefined age. MATERIAL AND METHODS: Karyotyping, fluorescence in situ hybridization, short tandem repeats and microarrays were used, depending on the type of loss and available sample. RESULTS: In 29 POCs we found maternal tissue and were eliminated from the analyses. Informative results were obtained in 250 (91.2 %)/274 cases; the karyotyping success rate was 80.7%; that of single nucleotide polymorphism microarrays, 94.5%; and that of fluorescence in situ hybridization and short tandem repeat, 100%. Cytogenetic abnormalities were observed in 57.6% of miscarriages and in 24.5% of stillbirths; 94% of total anomalies were numerical and 6% were submicroscopic. CONCLUSIONS: Karyotyping with simultaneous short tandem repeat study to rule out contamination of maternal cells is effective for studying miscarriages; in stillbirths, microarrays are recommended.

ANTECEDENTES: Las alteraciones cromosómicas están presentes en 50 a 60 % de los abortos espontáneos y en 6 a 19 % de los mortinatos. Aunque se prefieren los microarreglos para estudiarlos, numerosos hospitales no pueden ofrecerlos. OBJETIVO: Presentar los resultados del estudio citogenético de 303 productos de la concepción (POC), 184 se obtuvieron de abortos espontáneos, 49 fueron mortinatos y en 17 no se identificó la de edad gestacional. MATERIAL Y MÉTODOS: Se empleó cariotipo, hibridación in situ con fluorescencia, secuencias cortas repetidas en tándem y microarreglos, según el tipo de pérdida y la muestra disponible. RESULTADOS: En 29 POC se encontró tejido materno, por lo que fueron eliminados de los análisis. En 250 (91.2 %)/274 casos se obtuvieron resultados informativos; la tasa de éxito del cariotipo fue de 80.7 %; la de los microarreglos de SNP, de 94.5 %; y la de la hibridación fluorescente in situ y la repetición corta en tándem, de 100 %. Se observaron anomalías citogenéticas en 57.6 % de los abortos espontáneos y en 24.5 % de los mortinatos; 94 % de las anomalías fueron numéricas y 6 %, submicroscópicas. CONCLUSIONES: El cariotipo en conjunto con el estudio de secuencias cortas repetidas en tándem para descartar contaminación de células maternas es efectivo para estudiar abortos espontáneos; los microarreglos se recomiendan en los mortinatos.

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.

Estudio cromosómico de 303 pérdidas gestacionales en México / Chromosome analysis of 303 pregnancy losses in Mexico

Resumen Antecedentes: Las alteraciones cromosómicas están presentes en 50 a 60 % de los abortos espontáneos y en 6 a 19 % de los mortinatos. Aunque se prefieren los microarreglos para estudiarlos, numerosos hospitales no pueden ofrecerlos. Objetivo: Presentar los resultados del estudio citogenético de 303 productos de la concepción (POC), 184 se obtuvieron de abortos espontáneos, 49 fueron mortinatos y en 17 no se identificó la de edad gestacional. Material y métodos: Se empleó cariotipo, hibridación in situ con fluorescencia, secuencias cortas repetidas en tándem y microarreglos, según el tipo de pérdida y la muestra disponible. Resultados: En 29 POC se encontró tejido materno, por lo que fueron eliminados de los análisis. En 250 (91.2 %)/274 casos se obtuvieron resultados informativos; la tasa de éxito del cariotipo fue de 80.7 %; la de los microarreglos de SNP, de 94.5 %; y la de la hibridación fluorescente in situ y la repetición corta en tándem, de 100 %. Se observaron anomalías citogenéticas en 57.6 % de los abortos espontáneos y en 24.5 % de los mortinatos; 94 % de las anomalías fueron numéricas y 6 %, submicroscópicas. Conclusiones: El cariotipo en conjunto con el estudio de secuencias cortas repetidas en tándem para descartar contaminación de células maternas es efectivo para estudiar abortos espontáneos; los microarreglos se recomiendan en los mortinatos.

Abstract Background: Chromosomal abnormalities are present in 50 to 60 % of miscarriages and in 6 to 19 % of stillbirths. Although microarrays are preferred for studying chromosomal abnormalities, many hospitals cannot offer this methodology. Objective: To present the results of the cytogenetic analysis of 303 products of conception (POC), which included 184 miscarriages, 49 stillbirths and 17 cases of undefined age. Material and methods: Karyotyping, fluorescence in situ hybridization, short tandem repeats and microarrays were used, depending on the type of loss and available sample. Results: In 29 POCs we found maternal tissue and were eliminated from the analyses. Informative results were obtained in 250 (91.2 %)/274 cases; the karyotyping success rate was 80.7 %; that of single nucleotide polymorphism microarrays, 94.5 %; and that of fluorescence in situ hybridization and short tandem repeat, 100 %. Cytogenetic abnormalities were observed in 57.6 % of miscarriages and in 24.5 % of stillbirths; 94 % of total anomalies were numerical and 6 % were submicroscopic. Conclusions: Karyotyping with simultaneous short tandem repeat study to rule out contamination of maternal cells is effective for studying miscarriages; in stillbirths, microarrays are recommended.

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.

Serum sclerostin is associated with recurrent kidney stone formation independent of hypercalciuria.

Background: Kidney stones are frequent in industrialized countries with a lifetime risk of 10 to 15%. A high percentage of individuals experience recurrence. Calcium-containing stones account for more than 80% of kidney stones. Diet, environmental factors, behavior, and genetic variants contribute to the development of kidney stones. Osteocytes excrete the 21 kDa glycoprotein sclerostin, which inhibits bone formation by osteoblasts. Animal data suggests that sclerostin might directly or indirectly regulate calcium excretion via the kidney. As hypercalciuria is one of the most relevant risk factors for kidney stones, sclerostin might possess pathogenic relevance in nephrolithiasis. Methods: We performed a prospective cross-sectional observational controlled study in 150 recurrent kidney stone formers (rKSF) to analyse the association of sclerostin with known stone risk factors and important modulators of calcium-phosphate metabolism. Serum sclerostin levels were determined at the first visit. As controls, we used 388 non-stone formers from a large Swiss epidemiological cohort. Results: Sclerostin was mildly increased in rKSF in comparison to controls. This finding was more pronounced in women compared to men. Logistic regression indicated an association of serum sclerostin with rKSF status. In hypercalciuric individuals, sclerostin levels were not different from normocalciuric patients. In Spearman correlation analysis we found a positive correlation between sclerostin, age, and BMI and a negative correlation with eGFR. There was a weak correlation with iPTH and intact FGF 23. In contrast, serum sclerostin levels were not associated with 25-OH Vitamin D3, 1,25-dihydroxy-Vitamin D3, urinary calcium and phosphate or other urinary lithogenic risk factors. Conclusion: This is the first prospective controlled study investigating serum sclerostin in rKSF. Sclerostin levels were increased in rKSF independent of hypercalciuria and significantly associated with the status as rKSF. It appears that mechanisms other than hypercalciuria may be involved and thus further studies are required to elucidate underlying pathways.

A novel method for automated crystal visualization and quantification in murine folic acid-induced acute kidney injury.

Folic acid (FA)-induced acute kidney injury (FA-AKI) is an increasingly prevalent rodent disease model involving the injection of a high dose of FA that culminates in renal FA crystal deposition and injury. However, the literature characterizing the FA-AKI model is sparse and dated in part due to the absence of a well-described methodology for the visualization and quantification of renal FA crystals. Using widely available materials and tools, we developed a straightforward and crystal-preserving histological protocol that can be coupled with automated imaging for renal FA crystal visualization and generated an automated macro for downstream crystal content quantification. The applicability of the method was demonstrated by characterizing the model in male and female C57BL6/JRj mice after 3 and 30 h of FA treatment. Kidneys from both sexes and timepoints showed a bimodal distribution of FA crystal deposition in the cortical and medullary regions while, compared with males, females exhibited higher renal FA crystal content at the 30-h timepoint accompanied by greater kidney weight and higher plasma urea. Despite comparable plasma phosphate concentrations, FA-AKI resulted in a substantially more elevated plasma intact fibroblast growth factor 23 (FGF23) in females, reflected by a similar pattern in osseous Fgf23 mRNA expression. Therefore, the presented method constitutes a valuable tool for the quantification of renal FA crystals, which can aid the mechanistic characterization of the FA-AKI model and serves as a means to control for confounding changes in FA crystallization when using the model for investigating early and prophylactic AKI therapeutic interventions.NEW & NOTEWORTHY Here, we describe a novel method for the visualization and quantification of renal folic acid (FA) crystals in the rodent FA-induced acute kidney injury (FA-AKI) model. The protocol involves a straightforward histological approach followed by fully automated imaging and quantification steps. Applicability was confirmed by showing that the FA-AKI model is sex-dependent. The method can serve as a tool to aid in characterizing FA-AKI and to control for studies investigating prophylactic therapeutic avenues using FA-AKI.

Intact FGF23 predicts serum phosphate improvement after combined nicotinamide and phosphate binder treatment in hemodialysis patients.

Background: Hyperphosphatemia is associated with increased mortality and cardiovascular morbidity of end-stage kidney failure (ESKF) patients. Managing serum phosphate in ESKF patients is challenging and mostly based on limiting intestinal phosphate absorption with low phosphate diets and phosphate binders (PB). In a multi-centric, double-blinded, placebo-controlled study cohort of maintenance hemodialysis patients with hyperphosphatemia, we demonstrated the efficacy of nicotinamide modified release (NAMR) formulation treatment in addition to standard PB therapy in decreasing serum phosphate. Here we aimed to assess the relationship between phosphate, FGF23, inflammation and iron metabolism in this cohort. Methods: We measured the plasma concentrations of intact fibroblast growth factor 23 (iFGF23) and selected proinflammatory cytokines at baseline and Week 12 after initiating treatment. Results: We observed a strong correlation between iFGF23 and cFGF23 (C-terminal fragment plus iFGF23). We identified iFGF23 as a better predictor of changes in serum phosphate induced by NAMR and PB treatment compared with cFGF23. Recursive partitioning revealed at baseline and Week 12, that iFGF23 and cFGF23 together with T50 propensity were the most important predictors of serum phosphate, whereas intact parathyroid hormone (iPTH) played a minor role in this model. Furthermore, we found serum phosphate and iPTH as the best predictors of iFGF23 and cFGF23. Sex, age, body mass index, and markers of inflammation and iron metabolism had only a minor impact in predicting FGF23. Conclusion: Lowering serum phosphate in ESKF patients may depend highly on iFGF23 which is correlated to cFGF23 levels. Serum phosphate was the most important predictor of plasma FGF23 in this ESKF cohort.

Ovarian cancer G protein-coupled receptor 1 deficiency exacerbates crystal deposition and kidney injury in oxalate nephropathy in female mice.

Ovarian cancer G protein-coupled receptor 1 (OGR1) (Gpr68) and G protein-coupled receptor 4 (GPR4) (Gpr4) are proton-activated G protein-coupled receptors that are stimulated upon increased extracellular acidity. These receptors have various physiological and pathophysiological roles in renal acid-base physiology, tissue inflammation, and fibrosis among others. Their function in injured renal tissue, however, remains mostly unclear. To address this, we investigated their role in crystalline nephropathy by increasing the oxalate intake of GPR4 KO and OGR1 KO mice. After 10 days of high-oxalate intake and 4 days of recovery, renal crystal content, histopathology, filtration function, and inflammation were assessed. While GPR4 deficiency did not show major alterations in disease progression, OGR1 KO mice had higher urinary calcium levels and exacerbated crystal accumulation accompanied by decreased creatinine clearance and urea excretion and a decreased presence of regulatory T (Treg) cells in kidney tissue. When lowering the severity of the kidney injury, OGR1 KO mice were more prone to develop crystalline nephropathy. In this setting, OGR1 KO mice displayed an increased activation of the immune system and a higher production of proinflammatory cytokines by T cells and macrophages. Taken together, in the acute setting of oxalate-induced nephropathy, the lack of the proton-activated G protein-coupled receptor (GPCR) GPR4 does not influence disease. OGR1 deficiency, however, increases crystal deposition leading to impaired kidney function. Thus, OGR1 may be important to limit kidney crystal deposition, which might subsequently be relevant for the pathophysiology of oxalate kidney stones or other crystallopathies.

Blocking FGF23 signaling improves the growth plate of mice with X-linked hypophosphatemia.

Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone. X-linked hypophosphatemia (XLH) is the most prevalent inherited phosphate wasting disorder due to mutations in the PHEX gene, which cause elevated circulating FGF23 levels. Clinically, it is characterized by growth impairment and defective mineralization of bones and teeth. Treatment of XLH is challenging. Since 2018, neutralizing antibodies against FGF23 have dramatically improved the therapy of XLH patients, although not all patients fully respond to the treatment, and it is very costly. C-terminal fragments of FGF23 have recently emerged as blockers of intact FGF23 signaling. Here, we analyzed the effect on growth and bone of a short 26 residues long C-terminal FGF23 (cFGF23) fragment and two N-acetylated and C-amidated cFGF23 peptides using young XLH mice (Phex C733RMhda mice). Although no major changes in blood parameters were observed after 7 days of treatment with these peptides, bone length and growth plate structure improved. The modified peptides accelerated the growth rate probably by improving growth plate structure and dynamics. The processes of chondrocyte proliferation, death, hypertrophy, and the cartilaginous composition in the growth plate were partially improved in young treated XLH mice. In conclusion, these findings contribute to understand the role of FGF23 signaling in growth plate metabolism and show that this may occur despite continuous hypophosphatemia.

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.

Alkali therapy protects renal function, suppresses inflammation, and improves cellular metabolism in kidney disease.

Chronic kidney disease (CKD) affects approximately 10-13% of the population worldwide and halting its progression is a major clinical challenge. Metabolic acidosis is both a consequence and a possible driver of CKD progression. Alkali therapy counteracts these effects in CKD patients, but underlying mechanisms remain incompletely understood. Here we show that bicarbonate supplementation protected renal function in a murine CKD model induced by an oxalate-rich diet. Alkali therapy had no effect on the aldosterone-endothelin axis but promoted levels of the anti-aging protein klotho; moreover, it suppressed adhesion molecules required for immune cell invasion along with reducing T-helper cell and inflammatory monocyte invasion. Comparing transcriptomes from the murine crystallopathy model and from human biopsies of kidney transplant recipients (KTRs) suffering from acidosis with or without alkali therapy unveils parallel transcriptome responses mainly associated with lipid metabolism and oxidoreductase activity. Our data reveal novel pathways associated with acidosis in kidney disease and sensitive to alkali therapy and identifies potential targets through which alkali therapy may act on CKD and that may be amenable for more targeted therapies.

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.

Chronic High Phosphate Intake in Mice Affects Macronutrient Utilization and Body Composition.

SCOPE: In the last decades, dietary phosphate intake has increased due to a higher consumption of ultraprocessed food. This higher intake has an impact on body composition and health state. Recently, this study finds that a high chronic phosphate diet leads to no major renal alterations, but negatively affects parameters of bone health probably due to the chronic acid load. Here the effect of high phosphate consumption on parameters of energy metabolism is assessed. METHODS AND RESULTS: Healthy mature adult mice are fed for 1 year or 4 months with either a standard (0.6 % w/w) or a high phosphate (1.2 % w/w) diet. Males and females of two different genetic backgrounds are investigated. Mice feed the high phosphate diet show an attenuated body-weight gain, lower respiratory exchange ratio, decreased body fat mass, and increased lean-to-fat mass ratio. Moreover, the high phosphate diet leads to fasting hypoglycemia with no differences in the glucose response to an oral glucose tolerance test. Triglycerides and cholesterol in blood are similar independently of dietary phosphate content. However, 1-methylhistidine is lower in animals feed a chronic high phosphate intake. CONCLUSIONS: High phosphate diet attenuates body weight gain, but induces hypoglycemia and may alter muscle homeostasis.

Constitutive depletion of Slc34a2/NaPi-IIb in rats causes perinatal mortality.

Absorption of dietary phosphate (Pi) across intestinal epithelia is a regulated process mediated by transcellular and paracellular pathways. Although hyperphosphatemia is a risk factor for the development of cardiovascular disease, the amount of ingested Pi in a typical Western diet is above physiological needs. While blocking intestinal absorption has been suggested as a therapeutic approach to prevent hyperphosphatemia, a complete picture regarding the identity and regulation of the mechanism(s) responsible for intestinal absorption of Pi is missing. The Na+/Pi cotransporter NaPi-IIb is a secondary active transporter encoded by the Slc34a2 gene. This transporter has a wide tissue distribution and within the intestinal tract is located at the apical membrane of epithelial cells. Based on mouse models deficient in NaPi-IIb, this cotransporter is assumed to mediate the bulk of active intestinal absorption of Pi. However, whether or not this is also applicable to humans is unknown, since human patients with inactivating mutations in SLC34A2 have not been reported to suffer from Pi depletion. Thus, mice may not be the most appropriate experimental model for the translation of intestinal Pi handling to humans. Here, we describe the generation of a rat model with Crispr/Cas-driven constitutive depletion of Slc34a2. Slc34a2 heterozygous rats were indistinguishable from wild type animals under standard dietary conditions as well as upon 3 days feeding on low Pi. However, unlike in humans, homozygosity resulted in perinatal lethality.

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.

Evaluation of Placental Micro-vascularization by Superb Micro-vascular Imaging Doppler in Cases of Intra-uterine Growth Restriction: A First Step.

Superb micro-vascular imaging (SMI) Doppler has proven to be a valid method to assess normal placental micro-vascularization. In this study, we present the application of SMI Doppler to assess placental micro-vascularization in cases of placental insufficiency. We observed fewer secondary and tertiary villi in cases of intra-uterine growth restriction, as well as a lower pulsatile index of secondary villi. The observations made in our study stress the diagnostic potential of SMI Doppler in placental insufficiency.

A chronic high phosphate intake in mice is detrimental for bone health without major renal alterations.

BACKGROUND: Phosphate intake has increased in the last decades due to a higher consumption of processed foods. This higher intake is detrimental for patients with chronic kidney disease, increasing mortality and cardiovascular disease risk and accelerating kidney dysfunction. Whether a chronic high phosphate diet is also detrimental for the healthy population is still under debate. METHODS: We fed healthy mature adult mice over a period of one year with either a high (1.2% w/w) or a standard (0.6% w/w) phosphate diet, and investigated the impact of a high phosphate diet on mineral homeostasis, kidney function and bone health. RESULTS: The high phosphate diet increased plasma phosphate, parathyroid hormone (PTH) and calcitriol levels, with no change in fibroblast growth factor 23 levels. Urinary phosphate, calcium and ammonium excretion were increased. Measured glomerular filtration rate was apparently unaffected, while blood urea was lower and urea clearance was higher in animals fed the high phosphate diet. No change was observed in plasma creatinine levels. Blood and urinary pH were more acidic paralleled by higher bone resorption observed in animals fed a high phosphate diet. Total and cortical bone mineral density was lower in animals fed a high phosphate diet and this effect is independent of the higher PTH levels observed. CONCLUSIONS: A chronic high phosphate intake did not cause major renal alterations, but affected negatively bone health, increasing bone resorption and decreasing bone mineral density.

Systemic Jak1 activation provokes hepatic inflammation and imbalanced FGF23 production and cleavage.

Fibroblast growth factor 23 (FGF23) is a main regulator of mineral homeostasis. Low and high circulating FGF23 levels are associated with bone, renal, cardiovascular diseases, and increased mortality. Understanding the factors and signaling pathways affecting FGF23 levels is crucial for the management of these diseases and their complications. Here, we show that activation of the Jak1/Stat3 signaling pathway leads to inflammation in liver and to an increase in hepatic FGF23 synthesis, a key hormone in mineral metabolism. This increased synthesis leads to massive C-terminal FGF23 circulating levels, the inactive C-terminal fragment, and increased intact FGF23 levels, the active form, resulting in imbalanced production and cleavage. Liver inflammation does not lead to activation of the calcineurin-NFAT pathway, and no signs of systemic inflammation could be observed. Despite the increase of active intact FGF23, excessive C-terminal FGF23 levels block the phosphaturic activity of FGF23. Therefore, kidney function and renal αKlotho expression are normal and no activation of the MAPK pathway was detected. In addition, activation of the Jak1/Stat3 signaling pathway leads to high calcitriol levels and low parathyroid hormone production. Thus, JAK1 is a central regulator of mineral homeostasis. Moreover, this study also shows that in order to assess the impact of high FGF23 levels on disease and kidney function, the source and the balance in FGF23 production and cleavage are critical.

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.