Low-Phosphate Meals Accompanied by a Minimum Dose of CaCO3 Downregulates Pro-inflammation by Reducing CKD-MBD Indicators and Triggers by Decreasing Dietary Phosphate Intake

High dietary phosphate intake and poor adherence to phosphate-binding-therapy elevate the risk of hyperphosphatemia in maintenance hemodialysis (HD; MHD) patients. Therefore, chronic kidney disease-related mineral and bone disorder (CKD-MBD) indicators increase; consequently, risks of CKD-MBDs and inflammation are elevated. This double-blind, randomized control trial intervention study was designed to investigate the possibility of reducing blood CKD-MBD indicators and modulating inflammatory indicators by consuming low-phosphate (LP) meals accompanied by a minimum dose of a calcium-based phosphate binder (CaCO3). MHD patients were recruited and randomly assigned to an LP meal group (LP group) or a control group. After initial data collection, blood collection, and dietary counseling, subjects were asked to consume a washout diet for 1 week. During the washout diet period, subjects consumed their usual diet but took 1 tablet of calcium carbonate (1CaCO3) as a phosphate binder with each meal. After the washout diet period, subjects in the LP group and control group respectively consumed LP meals and regular meals twice a day for 1 week. Meat in the LP meals was boiled before the regular cooking process, but meat in control meals was not. All meals were supplied by a central kitchen so that the contents of phosphate and other nutrients could be identified. In total, 40 MHD patients completed the study program. After 1 week of the dietary intervention, the blood Ca x P product and dietary phosphate had significantly decreased in the LP group compared to the control group (p<0.05). The LP group had significantly lower variations in dietary phosphate intake, blood calcium, Ca x P product, and tumor necrosis factor (TNF)-α than the control group by comparing differences between after the dietary intervention and the baseline (△after intervention - baseline, p<0.05). The increase in dietary phosphate intake (△3rd - 2nd dietary phosphate intake) augmented the increase in the TNF-α level by 6.24-fold (odds ratio [95% confidence interval]: 6.24 [1.12~34.92], p<0.05). These results highlighted the conclusion that LP meals accompanied by a minimum dose of CaCO3 downregulated pro-inflammation by reducing CKD-MBD indicators which was triggered by decreasing dietary phosphate intake.

Small Intestinal Phosphate Absorption: Novel Therapeutic Implications

<b><i>Background:</i></b> Chronic kidney disease (CKD) affects approximately 15% of adults in the USA. As CKD progresses, urinary phosphate excretion decreases and results in phosphate retention and, eventually, hyperphosphatemia. As hyperphosphatemia is associated with numerous adverse outcomes, including increased cardiovascular mortality, reduction in phosphorus concentrations is a guideline-recommended, established clinical practice. Dietary phosphate restriction, dialysis, and phosphate binders are currently the only options for phosphate management. However, many patients with hyperphosphatemia have phosphorus concentrations &#x3e;5.5 mg/dL, despite treatment. <b><i>Summary:</i></b> This review pre­sents recent advances in the understanding of intestinal phosphate absorption and therapeutic implications. Dietary phosphate is absorbed in the intestine through two distinct pathways, paracellular absorption and transcellular transport. Recent evidence indicates that the paracellular route accounts for 65–80% of total phosphate absorbed. Thus, the paracellular pathway is the dominant mechanism of phosphate absorption. Tenapanor is a first-in-class, non-phosphate binder that inhibits the sodium-hydrogen exchanger 3 or solute carrier family 9 member 3 (SLC9A3) encoded by the SLC9A3 gene, and blocks paracellular phosphate absorption. <b><i>Key Messages:</i></b> Targeted inhibition of sodium-hydrogen exchanger 3 effectively reduces paracellular permeability of phosphate. Novel therapies that target the paracellular pathway may improve phosphate control in chronic kidney disease.

Novel insights into pulmonary phosphate homeostasis and osteoclastogenesis emerge from the study of pulmonary alveolar microlithiasis.

Pulmonary alveolar microlithiasis (PAM) is an autosomal recessive lung disease caused by a deficiency in the pulmonary epithelial Npt2b sodium-phosphate co-transporter that results in accumulation of phosphate and formation of hydroxyapatite microliths in the alveolar space. The single cell transcriptomic analysis of a PAM lung explant showing a robust osteoclast gene signature in alveolar monocytes and the finding that calcium phosphate microliths contain a rich protein and lipid matrix that includes bone resorbing osteoclast enzymes suggested a role for osteoclast-like cells in the defense against microliths. While investigating the mechanisms of microlith clearance, we found that Npt2b modulates pulmonary phosphate homeostasis through effects on alternative phosphate transporter activity and alveolar osteoprotegerin, and that microliths induce osteoclast formation and activity in a receptor activator of nuclear factor-κB ligand (RANKL) and dietary phosphate dependent manner. This work reveals that Npt2b and pulmonary osteoclast-like cells play key roles in pulmonary homeostasis and suggest potential new therapeutic targets for the treatment of lung disease.

FC 018HIGH DIETARY PHOSPHATE INTAKE AND INTRA-CARDIAC SYNTHESIS OF FIBROBLAST GROWTH FACTOR 23 SYNERGISTICALLY WORSEN CARDIAC FUNCTION

Background and Aims Left ventricular hypertrophy (LVH) is a major complication of CKD and associates with increased levels of the phosphaturic hormone fibroblast growth factor (FGF) 23. FGF23 induces hypertrophic growth of cardiac myocytes in vitro and LVH in rodents, suggesting that FGF23 can directly affect the heart. Besides the bone, cardiac myocytes express FGF23, too, and recent studies demonstrate that its expression is increased in cardiac and kidney injury, suggesting that cardiotoxicity of FGF23 may be at least partly due to the paracrine effects of heart-derived FGF23. However, it is still questioned whether elevated FGF23 per se is able to induce pathologic alterations in the heart or whether additional factors in CKD, such as Klotho deficiency or hyperphosphatemia are required for FGF23 to tackle the heart. By generating a mouse model with cardiac-specific overexpression of FGF23 via myocardial gene transfer using adeno-associated virus (AAV), we elucidated the cardiotoxic properties of elevated FGF23 in (1) unchallenged mice, unbiased of alterations usually associated with CKD, and (2) in the presence of high dietary phosphate intake, mimicking the exposure of enhanced serum phosphate. Method First, an adeno-associated virus that expresses murine Fgf23 (AAV-Fgf23) under the control of the cardiac troponin T promotor was injected subcutaneously into eight-week-old male C57BL/6 wildtype mice. After four months, cardiac function and geometry was assessed by cardiac magnetic resonance imaging (MRI) and echocardiography and heart tissue was analysed by qPCR, immunoblot and histological analyses. The biological activity of AAV-Fgf23-derived cardiac Fgf23 was determined using isolated neonatal rat ventricular myocytes (NRVM) in vitro. Second, AAV-Fgf23 and control mice were fed a 2% high phosphate diet (HPD) or a 0.8% normal phosphate diet (NPD) and cardiac phenotype was investigated after six months. Results AAV-Fgf23 mice showed increased cardiac-specific Fgf23 expression and synthesis of intact Fgf23 (iFgf23) protein in the heart resulting in enhanced circulating iFgf23 compared to control. Serum of AAV-Fgf23 mice stimulated hypertrophic growth of isolated NRVM and induced pro-hypertrophic gene expression in vitro, indicating that cardiac iFgf23 is biologically active. Likewise, AAV-Fgf23 mice revealed an activation of renal FGFR1/Klotho/MAPK signalling and subsequent down-regulation of renal sodium phosphate transporters NaPi-2a and NaPi-2c, causing reduced tubular phosphate reabsorption. Nevertheless, in unchallenged AAV-Fgf23 mice, impaired cardiac function, LVH and LV fibrosis were lacking. In contrast, HPD stimulated the bone expression of Fgf23 in both AAV-Fgf23 and Ctrl mice, while intra-cardiac Fgf23 mRNA levels were only increased in both AAV-Fgf23 groups irrespective of NPD or HPD. However, HPD in AAV-Fgf23 mice promoted O-glycosylation of cardiac iFgf23, suggesting stabilization of biologically active Fgf23 protein. Echocardiography showed impaired cardiac function in AAV-Fgf23 on HPD compared to its NPD group, demonstrated by enhanced end-systolic and end-diastolic volumes, increased systolic and diastolic LV diameters as well as enlarged LV inner diameters, respectively. Pressure-volume analysis using Millar catheter showed higher end-systolic and end-diastolic blood pressure (ESP, EDP) in AAV-Fgf23 mice on HPD compared to NPD. HPD in Ctrl only enhanced EDP, although this did not reach the level of statistical significance. Conclusion Chronic exposure to biologically active cardiac iFgf23 per se does not tackle the heart, while high intra-cardiac Fgf23 synthesis in the presence of high dietary phosphate promotes cardiotoxicity of Fgf23, which could pose a significant health risk to the general population.

Low Phosphate Diet Exacerbates Hypercalciuria in the Jimbee Mouse Model of SGLT2 Loss-of Function

Selective sodium-dependent glucose co-transporter 2 inhibitors (SGLT2is) are a class of anti-hyperglycemic drugs that lower blood glucose in an insulin-independent manner by inhibiting renal glucose reabsorption and promoting glucosuria. In persons with chronic kidney disease, a potential therapeutic target group for such SGLT2i treatment, dietary phosphate restriction is a mainstay of treatment for metabolic bone disease. We investigated the impact of a low phosphate (LP) diet on the physiology of Jimbee mice which, via deletion in exon 10 of the sglt2 gene, provide a model of SGLT2 loss-of-function, albeit with otherwise normal renal function. Male (M) and female (F), 12-week (wk) old, C57BL/6J (genetic control) and Jimbee mice were randomized 1:1 to a kcal/g equivalent 0.1% phosphate (LP) or 0.4% phosphate (normal P = NP) diet and monitored for 12 wks (n=9–12 per group x 8 groups). At study end (~24 wks of age), male Jimbee vs. C57BL/6J mice had lower body mass (BM: p&lt;0.0001), more-so on LP diet (C57BL/6J vs. Jimbee; (M) NP: 31.4 ± 2.1 vs. 28.6 ± 2.0. LP: 30.8 ± 2.0 vs. 26.0 ± 1.6 g). Female mice exhibited no differences in BM. By MRI, male mice demonstrated proportionate decrements in body composition of Jimbees, as the % fat vs. lean mass and % total body water were comparable between genotypes. HbA1c and random blood glucose were no different between groups, while glucosuria was increased in M and F Jimbee mice (p&lt;0.0001) on either diet [C57BL/6J vs. Jimbee; (M) NP: 0.2 ± 0.2 vs. 10.2 ± 4.5. LP: 0.2 ± 0.2 vs. 7.8 ± 2.0 mg/g (body weight)/day. (F) NP: 0.5 ± 0.5 vs. 8.2 ± 2.7. LP: 0.4 ± 0.3 vs. 7.0 ± 2.9 mg/g/day]. Serum calcium and phosphorus were no different between any groups. However, Jimbee mice also exhibited hypercalciuria and hyperphosphaturia (p&lt;0.001 for both). Hypercalciuria was amplified by LP diet in both strains, with a significant diet x strain interaction in males (p=0.01) [C57BL/6J vs. Jimbee; (M) NP: 4.7 ± 2.3 vs. 15.5 ± 8.2. LP: 27.8 ± 31.5 vs. 73.4 ± 25.8 µg/g/day of urine calcium (Ca2+). (F) NP: 4.9 ± 2.8 vs. 22.7 ± 16.9. LP: 45.8 ± 29.5 vs. 62.6 ± 39.8 µg/g/day]. In contrast, hyperphosphaturia was attenuated by LP diet [C57BL/6J vs. Jimbee; (M) NP: 8.7 ± 8.5 vs. 14.7 ± 10.4. LP: 0.9 ± 0.5 vs. 3.2 ± 2.9 µg/g/day of urine phosphate (PO4). (F) NP: 4.4 ± 6.1 vs. 16.3 ± 9.7. LP: 1.2 ± 0.8 vs. 2.9 ± 1.0 µg/g/day]. Plasma PTH levels were significantly lower (p&lt;0.001) in male Jimbee mice on either diet (C57BL/6J vs. Jimbee; NP: 81.1 ± 31.0 vs. 41.3 ± 10.7. LP: 38.2 ± 1.9 vs. 24.1 ± 6.2 pg/mL) and negatively correlated with daily urine Ca2+ (r = -0.62; p=0.006). Consistent with PTH, renal 1-α hydroxylase gene expression was decreased by ~60% in Jimbee males, specifically on LP diet (p=0.02). Together, these data suggest that, in mice, dietary phosphate restriction might exacerbate SGLT2i-related hypercalciuria during prolonged treatment, independent of PTH, becoming potentially detrimental to bone mineralization and growth over time.

Phosphate, Microbiota and CKD

Phosphate is a key uremic toxin associated with adverse outcomes. As chronic kidney disease (CKD) progresses, the kidney capacity to excrete excess dietary phosphate decreases, triggering compensatory endocrine responses that drive CKD-mineral and bone disorder (CKD-MBD). Eventually, hyperphosphatemia develops, and low phosphate diet and phosphate binders are prescribed. Recent data have identified a potential role of the gut microbiota in mineral bone disorders. Thus, parathyroid hormone (PTH) only caused bone loss in mice whose microbiota was enriched in the Th17 cell-inducing taxa segmented filamentous bacteria. Furthermore, the microbiota was required for PTH to stimulate bone formation and increase bone mass, and this was dependent on bacterial production of the short-chain fatty acid butyrate. We review current knowledge on the relationship between phosphate, microbiota and CKD-MBD. Topics include microbial bioactive compounds of special interest in CKD, the impact of dietary phosphate and phosphate binders on the gut microbiota, the modulation of CKD-MBD by the microbiota and the potential therapeutic use of microbiota to treat CKD-MBD through the clinical translation of concepts from other fields of science such as the optimization of phosphorus utilization and the use of phosphate-accumulating organisms.

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.