Intestinal Phosphorus Absorption in Moderate CKD and Healthy Adults Determined Using a Radioisotopic Tracer

Abstract

Visual Abstract

Background 

Reducing intestinal phosphorus absorption is a cornerstone in CKD-MBD management. Yet, knowledge gaps include how CKD pathophysiology affects intestinal phosphorus absorption. In vivo rodent studies suggest that intestinal phosphorus absorption remains inappropriately normal in early-moderate CKD, despite declining 1,25-dihydroxyvitamin D (1,25D). We measured intestinal phosphorus absorption in patients with moderate CKD versus healthy adults using a direct radiotracer method.

Methods 

Patients with CKD and healthy adults matched for age, sex, and race were enrolled in this 8-day controlled diet study: the first 6 days outpatient and the final 2 days inpatient. Oral and intravenous doses of ³³P and serial blood and urine sampling determined intestinal phosphorus absorption during the final 2 days. Secondary outcomes included fasting biochemistries and 24-hour urine phosphorus (uP).

Results 

In total, n=8 patients with CKD (eGFR=29–55 ml/min per 1.73 m²) and n=8 matched healthy controls completed the study. On a controlled diet, no difference in fractional intestinal phosphorus absorption was detected between patients with CKD and healthy adults (0.69 versus 0.62, respectively; P=0.52), and this was similar for 24-hour uP (884 versus 935 mg/d, respectively; P=0.70). Fractional intestinal phosphorus absorption was not significantly related to 24-hour uP. Patients with CKD had higher serum intact PTH and intact FGF23 and lower 1,25D. The relationship between 1,25D and fractional intestinal phosphorus absorption was not statistically significant.

Conclusions 

Intestinal phosphorus absorption with typical dietary intake did not differ in patients with moderate CKD compared with controls, despite lower serum 1,25D levels. In this setting, a relationship between 24-hour uP and fractional or absolute intestinal absorption was not evident. Further investigation is needed to determine what factors influence intestinal phosphorus absorption in CKD and the apparent lack of compensation by the intestine to limit phosphorus absorption in the face of declining kidney function and reduced 1,25D. Whether this is evident across a range of dietary phosphorus intakes, as well as CKD severity, also needs to be determined.

Clinical Trial registry name and registration number: 

Phosphorus Absorption in Healthy Adults and in Patients with Moderate Chronic Kidney Disease, NCT03108222

Reducing intestinal phosphorus absorption is the focus of therapies for management and prevention of CKD-mineral bone disorder.¹ These therapies include dietary phosphorus restriction and/or phosphate binding medications,^2–4 along with numerous pharmaceuticals in development targeting intestinal phosphorus absorption.^5,6 However, factors controlling and influencing phosphorus absorption, including the effects of CKD pathophysiology, on phosphorus absorption are not well understood.

Part of this uncertainty is due to the existence of few in vivo intestinal phosphorus absorption studies in animals^7–9 or humans,^10,11 in either healthy subjects or subjects with CKD. The effect of moderate CKD on intestinal phosphorus absorption is unclear, but the decreased 1,25-dihydroxyvitamin D (1,25D) levels observed at these stages of disease¹² would suggest that intestinal phosphorus absorption should be lower in these patients compared with healthy persons. This is due to the effects of 1,25D established in vitro^13,14 to increase expression of intestinal brush border membrane phosphate transporters.

However, we¹⁵ and others¹⁶ have shown that intestinal phosphorus absorption is not lower in rat models of CKD using in vivo absorption assessment methods, despite reduced 1,25D. This has not yet been rigorously tested in human studies of patients with moderate CKD. We have recently shown¹⁷ that 24-hour urine phosphorus (uP; 24-hour uP) is not a reliable biomarker of intestinal phosphorus absorption in patients with moderate CKD in a secondary analysis of a controlled feeding metabolic balance study.¹⁸ Together, these data indicate the need for more direct assessment measures of intestinal phosphorus absorption in studies of patients with CKD. The aim of this study was to determine fractional intestinal phosphorus absorption in patients with moderate CKD compared with healthy adults using a direct radioisotopic method in the context of a controlled study diet.

The use of isotopic tracers to measure intestinal absorption of minerals has been long considered the gold standard.^10,11,19 This is due to the ability to determine the movement of the tracer from the intestine into the blood and then, mineral transport rates between physiologic compartments by using direct measures of known doses of isotopes.²⁰ No stable isotopes of phosphorus exist (beyond the abundant ³¹P), and there are only two useful radioisotopes: ³²P (a high-energy β-particle emitter) and ³³P (a low-energy β-particle emitter). Previous studies utilizing phosphorus radioisotopes have been in patients with ESKD and have used ³³P together with the high-energy ³²P, presenting more hazardous radiation dose to the subjects.²¹ We have previously used a single radioisotope, ⁴⁵Ca, to mimic a dual-isotopic method to successfully determine calcium absorption and kinetics in patients with moderate CKD by staggering the oral and intravenous tracer doses by a day.¹⁸ More recently, we have utilized this same approach for assessment of intestinal phosphorus absorption using only the single lower-energy ³³P radioisotope in a recently published study in patients on hemodialysis.²² Here, we apply the same method for measuring fractional intestinal phosphorus absorption in patients with moderate-stage CKD and healthy adults.

Methods

Study Design and Participants

This was a parallel-arm study designed to assess fractional intestinal phosphorus absorption in patients with moderate CKD and healthy adults. Inclusion criteria for subjects with moderate CKD included men and women aged between 30 and 75 years with eGFR of 45–59 ml/min per 1.73 m² (stage 3a CKD) with A2 or A3 albuminuria or eGFR of 30–44 ml/min per 1.73 m² (stage 3b CKD) with or without albuminuria. Participants were required to be stable on all medications for 6 weeks prior to study enrollment and were required to discontinue all medications and nutritional supplements that could alter phosphorus metabolism (including vitamin D supplements [ergo- or cholecalciferol], calcium supplements, multivitamin/mineral supplements, calcitriol or active vitamin D analogs, calcimimetics, parathyroid hormone [PTH] analogs, and phosphate binder medications). Healthy control subjects were recruited using a voluntary database maintained by the Indiana Clinical Translational Science Institute. Healthy controls were race, sex, and age matched (±10 years) to patients with CKD. Healthy participants were required to have normal kidney function as assessed by a study nephrologist and no presence of albuminuria. Healthy controls were required to have normal fasting serum calcium, phosphate, and PTH values. In total, n=8 patients with moderate CKD and n=8 healthy matched controls completed the study.

Enrolled subjects participated in an 8-day controlled feeding study. For days 1–6, subjects consumed a controlled diet as outpatients and on day 7, were admitted to the Indiana Clinical Research Center (CRC) as inpatients for 2 days for phosphorus absorption measurement (Figure 1). All study protocols were approved by the Indiana University Institutional Review Board (Institutional Review Board Protocol No. 1612460566), and subjects gave their written informed consent. This study was registered with Clinicaltrials.gov (NCT03108222).

Figure 1.: Intestinal phosphorus absorption study design and protocol. Subjects followed a controlled study diet for 6 days at home, with all food provided as meal packouts. On Day 7, subjects were admitted to the CRC and began the intestinal phosphorus absorption test protocol which included an oral dose of ³³P on Day 7 and an IV dose of ³³P on Day 8. Urine and sequential blood draws were collected for determination of intestinal phosphrous absorption by kinetic modeling. Subjects completed the study and were discharged from the CRC on the morning of Day 9. IV, intravenous.

Controlled Study Diet

The study diet consisted of a 3-day cycle menu designed by a registered dietitian bionutritionist using ProNutra dietary analysis software (Viocare, Inc., Princeton, NJ). All study meals (outpatient pack outs and inpatient meals) were prepared in a metabolic kitchen where individual ingredients were precisely measured to the 0.1 g. The cycle menu was designed as a high-phosphorus diet (approximately 1500 mg/d phosphorus), reflective of the average United States daily phosphorus intake.^23,24 Energy, protein, fiber (approximately 25 g/d), and other mineral (approximately 1100 mg/d calcium, approximately 2400 mg/d sodium, and approximately 3200 mg/d potassium) compositions were also controlled during the study period. Average kilocalories computed from ProNutra were 2240±15 kcal/d, and protein was 109±10 g/d. The diet was designed at one calorie level and to be consistent in nutrient content across the 3-day cycle menu.²⁵

Sources of dietary phosphorus were selected to include both plant (30% of total dietary protein) and animal (70% of total dietary protein) sources. Foods with inorganic phosphate additives were not included due to the high bioaccessibility of these additives.^23,26 Major forms of phosphorus in the diet included milk and other dairy products, meat (i.e., beef or poultry), and plant proteins, such as lentils or beans. The 3-day cycle menu details are included in Supplemental Tables 1 and 2. During the 2 days of inpatient phosphorus absorption testing, patients were given the same menus both days (cycle day 1) to limit the potential effect of different foods among the 3-day cycle menu on intestinal phosphorus absorption efficiency.

Actual mineral composition of homogenated daily diet composites for the 3-day cycle menu was determined by inductively coupled plasma-optical emission spectrometry (ICP-OES; Optima 4300DV; Perkin Elmer, Shelton, CT). Dietary compliance during the inpatient stay was assessed using weights from leftover food after meals, and dietary components were calculated as percentage of meal consumed.

Fractional Intestinal Phosphorus Absorption and Phosphorus Kinetics

The inpatient phosphorus absorption test included oral and intravenous doses of ³³P. On study day 7, patients arrived at CRC fasting, and blood sample and urine sample were taken upon admission to CRC. Following fasting blood and urine samples, an oral dose of 10 μCi of ³³P as orthophosphoric acid (Perkin Elmer, Waltham, MA [discontinued product] or American Radiolabeled Chemicals, Inc., St. Louis, MO) in 120 ml mineral-free water was administered to the patient halfway through the first meal of the day (breakfast) containing approximately 1/3 of the daily phosphorus intake. After the oral dose was administered, patients finished the remainder of their meal. Serial blood draws then occurred at the following time points from tracer ingestion: 15, 30, 45, 60, and 90 minutes and 2, 3, 4, 6, and 24 hours. Urine samples were pooled and collected during the following intervals postdosing: 0–2 hours postdose, 2–4 hours postdose, 4–6 hours postdose, 6–24 hours postdose, and 24–25 hours postdose. All inpatient fecal samples were collected, and times were recorded. At 25 hours after the oral dose, a blood sample was taken; then, subjects were administered an intravenous dose of 10 μCi of ³³P in sterile saline. Serial blood draws and urine and fecal collections after intravenous dose followed the same protocol as after oral dosing indicated above.

³³P activity in serum, urine, and feces was measured via liquid scintillation counting (Tri-Carb 2910 TR Liquid Scintillation Analyzer; Perkin Elmer). The primary outcome, fractional intestinal phosphorus absorption, was determined by multicompartment kinetic modeling using general equation solving software (WinSAAM). Absorption was calculated as the fraction of the tracer entering circulation relative to the total dose moving through the digestive system. Both oral and intravenous ³³P tracer doses are needed to model the movement of phosphorus from the gut into circulation (oral tracer) while taking into account the rate of removal of phosphorus from circulation (intravenous tracer). Staggering the oral and intravenous doses by a day allows for data collection of the oral dose data alone on the first day, followed by data collection reflecting the intravenous dose on the second day. This method is made possible because absorption is completed by approximately 7.5 hours after oral dosing (on the basis of the work of Farrington et al.¹⁰ from 1981 using dual-isotope ³³P and ³²P tracers), and the residual signal of the oral ³³P tracer in circulation prior to intravenous dosing on the second day is more than a full order of magnitude lower than what is detected following the intravenous ³³P tracer dose.²² Further details on the compartmental model are provided in Supplemental Figure 1.

Serum Biochemical Measures

Fasting biochemistries (serum calcium, phosphorus, and creatinine) at screening and study day 1 were analyzed by the Indiana University Pathology Laboratory by standard clinical chemistry for study eligibility and safety monitoring. Inpatient serum and urine samples were analyzed for calcium, phosphorus, and creatinine by clinical chemistry analyzer (RX Daytona; Randox Laboratories Ltd, Crumlin, United Kingdom). Fasting serum samples from study day 7 (inpatient) were collected and analyzed for phosphorus-regulating hormones. Serum 1,25D was measured by liquid chromatography with tandem mass spectrometry (Mayo Clinic Laboratories, Rochester, MN). Serum PTH was measured using the intact parathyroid hormone (iPTH) 1–84 ELISA kit (Alpco, Salem, NH). Serum fibroblast growth factor 23 (FGF23) was measured by a chemiluminescence enzyme immunoassay kit (MedFrontier intact fibroblast growth factor [iFGF23]; Eagle Biosciences, Amherst, NH).

Phosphorus Balance, uP, and Fecal Phosphorus

During the 48-hour inpatient stay, the controlled study diet was continued, and all feces and urine were collected. Subjects were provided with bottled mineral-free water and encouraged by nursing and study staff to consume a minimum of two 16–fluid ounce bottles per day up to a maximum of four bottles per day in addition to the food and beverages provided at meals to maintain adequate hydration. Urine collections were timed by research nursing staff who ensured that start and end times were accurate. At the end time for each urine collection, subjects were instructed to empty their bladder a final time in the collection container to end that time point. At the end of the time point, the container was taken to the laboratory, where research staff measured the volume in graduated cylinders and began processing the urine according to protocol.

Urine sample volumes were then measured by trained laboratory staff, aliquoted, and acidified with 1% (vol/vol) concentrated hydrogen chloride for storage at −80°C. Upon mineral analysis, aliquots were diluted with 2% nitric acid for analysis by ICP-OES.

The 24-hour uP was measured by ICP-OES for each of the inpatient study days. The 2-day average 24-hour uP from each 24-hour pooled collection was used in analysis. Fractional excretion of phosphorus (FE_Pi) was calculated by the following equation: [uP (milligrams per deciliter) × serum creatinine (milligrams per deciliter)]/[serum phosphorus (milligrams per deciliter) × urine creatinine (milligrams per deciliter)]×100.²⁷ Tubular maximum reabsorption of phosphorus (TmP/GFR) was calculated as [1−(uP (milligrams per deciliter) × serum creatinine (milligrams per deciliter)]/[serum phosphorus (milligrams per deciliter) × urine creatinine (milligrams per deciliter)]×100/eGFR. Fasting urine and serum phosphorus and creatinine from inpatient day 7 (or preoral ³³P dose) were used for these calculations.

Adequate dietary fiber²⁵ was provided by the diet with an average of 26 g/d (calculated by ProNutra software) with adequate fluids to aid gastric motility. Subjects were provided with collection containers for each fecal sample. Research nursing staff recorded the date and time on the collection container and flash froze the fecal samples on dry ice prior to transferring to a −20°C freezer for storage until analysis. Thawed fecal samples were homogenized and aliquoted into microwave digestion vessels with 5 ml of 70% nitric acid and 5 ml of ultrapure water. Fecal samples were digested in microwave (MARS 6; CEM, Matthews, NC) at 210°C for 35 minutes. Digested fecal samples were diluted with ultrapure water to 2% nitric acid for mineral analysis (Optima 4300DV; Perkin Elmer).¹⁸ Mineral content of feces was normalized to excretion of a nonabsorbable fecal maker, polyethylene glycol (PEG; molecular weight 3500), over 48 hours to account for loss of sample during collection and fecal transit time; 2.96±0.12 g/d PEG was taken as approximately 1 g with each meal during the study period. PEG concentration of fecal samples was measured by turbidimetric assay using spectrophotometry.²⁸

Phosphorus balance was determined using 2-day average values for the daily dietary phosphorus intake as an inpatient, 24-hour uP, and fecal phosphorus excretion normalized to fecal PEG excretion.¹⁷ Phosphorus balance values were calculated from the following equation: phosphorus balance (milligrams per day) = dietary phosphorus intake (milligrams per day) – urinary phosphorus excretion (milligrams per day) – fecal phosphorus excretion (milligrams per day).

Statistical Analyses

All statistical analyses were performed using SAS v9.4 (SAS Institute, Inc., Cary, NC) with statistical significance set at α=0.05. Descriptive statistics were performed for subject characteristics, baseline biochemical measures, and dietary compliance. Linear regression (procedure general linear model [PROC GLM]) was used to determine differences between patients with CKD and controls for the outcomes of fractional intestinal phosphorus absorption, absolute phosphorus absorption (milligrams per day), 24-hour uP, FE_Pi, TmP/GFR, phosphorus balance, serum 1,25D, serum iPTH, and serum iFGF23, taking matched pairs into account in the regression model. The relationships between phosphorus absorption (fractional and absolute) and 24-hour uP; fractional intestinal phosphorus absorption and serum 1,25D, iPTH, and iFGF23; and 24-hour uP, FE_Pi, and TmP/GFR with iPTH and iFGF23 were determined by Pearson correlations. All correlations were performed in all patients and within each group (CKD and healthy).

Power calculations to show noninferiority indicated a total sample size of n=16 (n=8 per group) to provide 80% power with α=0.05 to detect a fractional intestinal phosphorus absorption difference of 0.13 between groups with an SD of 0.10.^21,29

Results

Dietary Analysis and Subject Characteristics

Average dietary macronutrient (calculated from ProNutra software) and mineral composition (by ICP-OES) for the controlled diets is described in Table 1. The average value of phosphorus was 1828±101 mg/d. All mineral data presented are from inductively coupled plasma mineral analysis. Mineral composition values of the controlled diet inductively coupled plasma analysis differed from dietary software analysis (Supplemental Table 1). Adequate intake for dietary fiber was provided by the diet with an average of 25.5±0.3 g of dietary fiber per day (calculated by ProNutra software). Subjects had high dietary compliance (average of 94%): n=11 of 16 consumed 95%–100% of their meals, n=13 of 16 consumed >90%, n=2 consumed between 85% and 89%, and only n=1 had relatively poor dietary compliance at 68%. This participant’s fractional intestinal phosphorus absorption value was in the middle of the distribution of values. Further, dietary compliance was explored as a covariate and was not significant; therefore, it was not included in the analyses.

Table 1. - Average daily composition of prepared study diet

Dietary Component	Mean (SD)
Energy, kcal/d	2240 (15)
Protein, g/d	109 (10)
Carbohydrate, g/d	288 (6)
Fat, g/d	77 (3)
Phosphorus, mg/d	1828 (101)
Calcium, mg/d	884 (24)
Sodium, mg/d	2487 (178)
Potassium, mg/d	3121 (369)
Magnesium, mg/d	332 (32)

Subject characteristics and day 1 baseline biochemistries for each study group are described in Table 2. As expected, BUN and eGFR were significantly different between the healthy controls and patients with CKD. No other differences in baseline subject characteristics were observed.

Table 2. - Subject demographics and biochemistries

Baseline Characteristics	Healthy, n=8	CKD, n=8	P Value
Age, yr	52 (13)	57 (14)	0.54
Women:men, n	4:4	4:4
Black participants:White participants, n	3:5	3:5
BMI, kg/m2	28.4 (4.7)	31.6 (8.7)	0.41
Baseline, day 1 fasting measures
BUN, mg/dl	15 (4)	32 (13)	0.008
eGFR, ml/min per 1.72 m2	88 (12)	41 (8)	<0.001
Serum phosphorus, mg/dl	3.3 (0.4)	3.4 (0.6)	0.88
Serum calcium, mg/dl	9.6 (0.2	9.3 (0.3)	0.05
Preabsorption testing, day 7 fasting measures
Serum 1,25D, pg/ml	38 (10)	26 (10)	0.03
Serum iPTH, pg/ml	46 (14)	110 (40)	0.001
Serum iFGF23, pg/ml	34 (10)	89 (35)	0.005

Data are presented as mean (SD) except for race and sex, which are presented as n. BMI, body mass index.

Intestinal Phosphorus Absorption

There was no statistical difference in fractional or absolute intestinal phosphorus absorption between patients with CKD and healthy controls (Figure 2). Patients with CKD had a mean fractional intestinal phosphorus absorption of 0.69±0.06, and healthy controls had a mean fractional intestinal phosphorus absorption of 0.62±0.07 (P=0.52; mean difference, 0.064; 95% confidence interval [95% CI], −0.12 to 0.25). Similarly, total phosphorus (milligrams) absorption values for patients with CKD and healthy controls were 1100±106 and 1137±127 mg/d, respectively (mean difference, −38; 95% CI, −393 to 318; P=0.66). Mean whole-body phosphorus retention (over a 2-day period) was not statistically different between patients with CKD (149±144 mg/d) compared with healthy controls (108±144 mg/d; mean difference, 41; 95% CI, −492 to 573; P=0.85).

Figure 2.: Intestinal phosphorus absorption in patients with CKD and healthy adults. (A) fractional intestinal absorption in patients with CKD and healthy adults. (B) Absolute intestinal phosphorus absorption in milligrams per day. Black lines indicate the means, and error bars are SEMs; triangles indicate values for subjects with CKD, and circles indicate values for healthy subjects.

Twenty-Four–Hour Urinary Phosphorus Excretion, Fractional Phosphorus Excretion, and TmP

The 24-hour uP was 884±334 mg/d in patients with CKD compared with 935±134 mg/d in healthy controls (mean difference, −50; 95% CI, −12 to −23; P=0.70) (Figure 3A). FE_Pi was higher in patients with CKD (29%±4%) compared with healthy controls (12%±4%; mean difference, 17; 95% CI, 7 to 28; P=0.003) (Figure 3B). TmP/GFR was lower in patients with CKD (2.66±0.17) compared with healthy controls (3.22±0.17; mean difference, −0.57; 95% CI, −1.09 to −0.04; P=0.04). The 24-hour uP was not significantly correlated to fractional intestinal phosphorus absorption overall (r=0.13; 95% CI, −0.39 to 0.59; P=0.63) (Figure 3C) or within either group (CKD: r=0.17; 95% CI, −0.63 to 0.78; P=0.68; healthy: r=0.13; 95% CI, −0.63 to 0.77; P=0.75). Similarly, the relationship between 24-hour uP and the absolute (milligrams) amount of phosphorus absorbed was not statistically significant overall (r=0.42; 95% CI, −0.10 to 0.76; P=0.11) (Figure 3D) nor within either group (CKD: r=0.43; 95% CI, −0.40 to 0.87; P=0.30; healthy: r=0.52; 95% CI, −0.29 to 0.90; P=0.18).

Figure 3.: Measures of urinary phosphorus excretion and relationships with intestinal phosphorus absorption. (A) The 24-hour uP for each study group. (B) FE_Pi for each study group. (C) The relationship between 24-hour uP and fractional intestinal phosphorus absorption. (D) The relationship between 24-hour uP and absolute intestinal phosphorus absorption (milligrams per day). Black lines indicate the means, and error bars are SEMs; triangles indicate values for subjects with CKD, and circles indicate values for healthy subjects. Solid lines are the regression fit, shaded areas are the 95% confidence limits, and the dotted lines indicate the 95% prediction limits.

Phosphorus Regulatory Hormones

Patients with CKD had lower mean 1,25D compared with healthy controls (26±3.6 versus 38±3.7 pg/ml, respectively; mean difference, −12.4; 95% CI, −23 to −1; P=0.03) (Figure 4A). Patients with CKD had higher serum iPTH (110±14 pg/ml) compared with controls (46±5 pg/ml; mean difference, 64; 95% CI, 32 to 96; P=0.001) (Figure 4B) as well as higher iFGF23 (89±12 pg/ml) compared with controls (34±3 pg/ml; mean difference, 55; 95% CI, 27 to 83; P=0.005) (Figure 4C).

Figure 4.: Serum values of phosphorus regulatory hormones. (A) Serum 1,25D, (B) iPTH, and (C) iFGF23 in patients with CKD and healthy adults. Black lines indicate the means, and error bars are SEMs; triangles indicate values for subjects with CKD, and circles indicate values for healthy subjects.

The relationship between serum 1,25D and fractional intestinal phosphorus absorption was not statistically significant (r=−0.16; 95% CI, −0.61 to 0.37; P=0.56) (Figure 5A). Neither serum iPTH nor serum iFGF23 were significantly correlated with fractional intestinal phosphorus absorption (r=0.20; 95% CI, −0.33 to 0.63; P=0.46 [Figure 5B] and r=0.15; 95% CI, −0.38 to 0.60; P=0.58, respectively [Figure 5C]). Similarly, serum iPTH and iFGF23 were not significantly related to 24-hour uP (r=0.07; 95% CI, −0.54 to 0.44; P=0.81 [Figure 6A] and r=0.04; 95% CI, −0.53 to 0.46; P=0.88 [Figure 6B], respectively). However, FE_Pi was positively correlated with serum iPTH (r=0.61; 95% CI, 0.16 to 0.85; P=0.01) (Figure 6C) and iFGF23 (r= 0.72; 95% CI, 0.34 to 0.90; P=0.002) (Figure 6D), and TmP/GFR was negatively correlated with iPTH (r=−0.77; 95% CI, −0.91 to −0.44; P<0.001) (Figure 6E). iFGF23 did not reach significance (r=−0.31; 95% CI, −0.70 to 0.22; P=0.24) (Figure 6F).

Figure 5.: The relationship between phosphorus regulatory hormones and fractional intestinal phosphorus absorption. (A) The relationship between 1,25D and fractional phosphorus absorption. (B) The relationship between serum iPTH and fractional phosphorus absorption. (C) The relationship between serum iFGF23 and fractional phosphorus absorption. Solid lines are the regression fits, shaded areas are the 95% confidence limits, and the dotted lines indicate the 95% prediction limits. Triangles indicate values for subjects with CKD, and circles indicate values for healthy subjects.

Figure 6.: The relationship between phosphaturic hormones and urinary phosphorus excretion. (A) The relationship between 24-hour uP and serum iFGF23. (B) The relationship between FE_Pi and serum iFGF23. (C) The relationship between 24-hour uP and serum iPTH. (D) The relationship between FE_Pi and serum iPTH. (E) The relationship between TmP/GFR and serum iPTH. (F) The relationship between TmP/GFR and serum iFGF23. Solid lines are the regression fits, shaded areas are the 95% confidence limits, and the dotted lines indicate the 95% prediction limits. Triangles indicate values for subjects with CKD, and circles indicate values for healthy subjects.

Discussion

Our results show that fractional intestinal phosphorus absorption in patients with moderate-stage CKD is not detectably reduced compared with healthy control subjects matched for age, sex, and race in the context of a controlled feeding study while consuming a dietary phosphorus intake typical of the general population. This was despite lower serum 1,25D and higher iPTH and iFGF23 in patients with CKD, which is consistent with previously described biochemical alterations with kidney function decline.¹² Particularly, lower 1,25D should lead to decreased phosphorus absorption via decreased sodium-dependent phosphate transport according to classic mechanistic understanding¹⁴ largely on the basis of in vitro/ex vivo absorption assessment methods. However, emerging data from in vivo/in situ assessments of intestinal phosphorus absorption are complicating our understanding of intestinal phosphorus absorption. We have recently shown using in situ intestinal ligated loop absorption methods that phosphorus absorption was not lower in CKD rats (Cy/+ model) compared with healthy normal rats, despite lower 1,25D levels in the CKD rats.¹⁵ This matched the prior work by Marks et al.,¹⁶ who also showed no difference in intestinal phosphorus absorption by intestinal ligated loop method in 5/6th nephrectomized rats compared with sham-operated controls—again, despite lower 1,25D in the nephrectomized rats. Our study of intestinal phosphorus absorption efficiency in humans further supports those previous findings in two different CKD rat models. Further, in our previous rat study,¹⁵ we noted a peculiar finding—that fractional intestinal phosphorus absorption was statistically higher in CKD versus normal rats despite the lower 1,25D. In this human study, we again see (numerically but not statistically) slightly higher fractional intestinal phosphorus absorption in patients with CKD compared with healthy controls. Marks et al.¹⁶ also showed, numerically but not statistically, slightly higher fractional intestinal phosphorus absorption in the nephrectomized versus sham rats. These observations may well be spurious (n=34 pairs would have been needed in this study to produce a significant P value), but the potential maladaptation of increasing fractional intestinal phosphorus absorption in CKD in the context of declining 1,25D may warrant further investigation.

If confirmed across various dietary phosphorus intake and stages of CKD, the lack of relationship between serum 1,25D levels and phosphorus absorption may also suggest that observations of increased serum phosphorus with calcitriol or analog administration³⁰ may be due to bone or kidney effects rather than intestinal absorption. This needs to be tested in clinical studies and particularly, for patients with later-stage CKD who may be on a dietary phosphorus restriction in addition to receiving exogenous calcitriol or analog because it has been shown through vitamin D receptor knockout studies that dietary phosphorus restriction increases active phosphate transport independent of the actions of 1,25D.³¹ An additional consideration is that circulating 1,25D may not correspond with tissue levels of 1,25D as some studies have shown.^32,33 Thus, this is a possible confounding factor in attempting to capture the relationship between 1,25D and fractional intestinal phosphorus absorption.

The use of a single ³³P radioisotope given orally and intravenously provides a direct measure of phosphorus absorption for use in clinical studies. Historically, radioisotope protocols for investigating intestinal phosphorus absorption have used both the high-energy ³²P isotope and the lower-energy ³³P isotope in a classic dual-isotope method where both isotopes can be given concurrently (one orally and one intravenously) and distinguished in the biologic samples due to their different energy peaks by liquid scintillation counting.^{19,34 32}P, however, is a high-energy β-particle emitter that results in a greater effective dose of radiation to the body’s tissues compared with ³³P, and so, it is potentially more hazardous. ³³P also has a research advantage due to its longer t_1/2 (25.3 days) compared with ³²P (14.3 days), which makes it detectable in samples for longer periods of time. Thus, we modified the classic dual-isotope absorption method by giving ³³P as both the oral and intravenous dose, separated by 1 day, to take advantage of the longer t_1/2 of ³³P and to reduce the effective dose of radiation to the patients. This is similar to the calcium absorption protocol we published previously using ⁴⁵Ca isotope for both oral and intravenous doses separated by 1 day.¹⁸ The oral ³³P data are captured within the first 24 hours; then, another blood sample is taken prior to the intravenous ³³P administration, which concludes the oral dose curve. After the intravenous ³³P dose is administered, the radioactivity counts that appear in the serum are an order of magnitude higher than the final serum samples of the oral dose curve, rendering negligible any remaining radioactivity signal from the oral dose. The subsequent data through 48 hours are, therefore, regarded as clearance of the intravenous ³³P infusion for use in the multicompartment kinetic model to estimate fractional intestinal phosphorus absorption. Direct measures of intestinal phosphorus absorption, such as the dual-isotope method or the modified single-isotope method described here and in our previous work,²² are useful for physiologic studies of intestinal phosphorus absorption in patients with CKD. We have previously reported that the traditional biomarker of phosphorus absorption, 24-hour uP excretion, was not related to net phosphorus absorption determined by metabolic balance studies.¹⁷ We re-examined this relationship in this study and again show no significant association between 24-hour uP and fractional intestinal phosphorus absorption by radiotracer method at the level of dietary phosphorus intake studied. As we have stated previously,¹⁷ these analyses do not preclude the use of 24-hour uP as a proxy of intestinal phosphorus absorption in the context of randomized, controlled trials where the intervention has a known or suspected effect on intestinal absorption (e.g., phosphate binder trials) and cause and effect can be assumed. Rather, caution should be taken in interpretation of 24-hour uP from cross-sectional analyses or from intervention studies where there is no known or suspected effect on intestinal phosphorus absorption or where an effect on phosphorus retention is plausible.

The 24-hour uP values observed in patients with moderate-stage CKD in this study are consistent with previous literature for this population.¹² However, mean 24-hour uP was not detectably different between patients with CKD and healthy controls, even though the phosphaturic hormones PTH and FGF23 were both elevated (as expected) in the patients with CKD. A limitation in detecting differences in 24-hour uP may be error in collection, although this was minimized in this controlled research environment, where start and end times were closely implemented by research nursing staff, including ending a collection by always asking the subjects to empty their bladders at the scheduled end time. Further, FE_Pi was higher and TmP/GFR was lower in the patients with CKD compared with healthy controls, which is consistent with the known phosphaturic effects of FGF23 and PTH on remaining functional nephrons to increase fractional phosphorus excretion and decrease the TmP.^12,27,35

A limitation of our study is the inclusion of only one relatively high dietary phosphorus level. We chose a relatively high intake level reflective of a typical United State high-phosphorus diet^23,24 to be translational to real-world patients. An analysis of the National Health and Nutrition Examination Survey shows a usual phosphorus intake of approximately 1500–1700 mg/d in adult men aged 31–70 years and approximately 1100–1200 mg/d in women the same age.^36,37 However, classic understanding of transcellular versus paracellular absorption pathway contributions suggests that paracellular absorption predominates at high dietary phosphorus intakes, which would be expected to minimize the effect of 1,25D affecting the active, transcellular absorption pathway.^38,39 Thus, it is possible that a relationship between 1,25D status and fractional intestinal phosphorus absorption would be detectable with very low phosphorus intakes. However, this notion is not supported by our previous rat studies in both healthy Sprague–Dawley rats⁸ and the CKD Cy/+ rat model¹⁵ that used a very low phosphate concentration ([0.1 mM]) for the absorption testing by in vivo methods (jejunal ligated loop) and yielded similar conclusions.⁸ Marks et al.¹⁶ also showed no effect of low 1,25D on intestinal phosphorus absorption in 5/6th nephrectomized rats (also using in vivo jejunal ligated loops and a very low phosphate concentration of [0.1 mM]). Further, in our in vivo absorption study in rats with CKD,¹⁵ we found that sodium-dependent phosphorus absorption only accounted for approximately 30% of total phosphorus absorption even at that low luminal phosphate concentration of [0.1 mM]. This is in agreement with another detailed study by Marks et al.⁷ that showed a relatively stable proportion of sodium-dependent (transcellular) versus sodium-independent (paracellular) absorption of approximately 30%:70%, respectively, across a wide range of luminal phosphate concentrations from very low ([0.1 mM]) to quite high ([10 mM]), which was in contrast to the proportions they observed using the in vitro brush border membrane vesicle uptake assay across the same range of phosphate concentrations. It is the in vitro studies upon which the field has largely based our concepts of transcellular versus paracellular phosphorus absorption contributions, which may not be accurate in vivo. We surmise that the findings of Marks et al.⁷ may suggest that paracellular absorption could be the predominant pathway in vivo across what would be considered a reasonable range of low (e.g., 700 mg/d) to high (e.g., 2000+ mg/d) phosphorus intakes in humans. This concept requires further study in humans and animal models using in vivo direct methods for assessing intestinal phosphorus absorption over a range of dietary phosphorus intakes. Nevertheless, the high dietary phosphorus intake selected for this study is a limitation as, on the basis of the animal studies, this is a level where paracellular absorption would be expected to dominate and the effects of 1,25D would be minimized.

An additional limitation related to the study diet is that we do not know how the phosphorus intake level of the study diet compared with these particular subjects’ habitual diets. Fractional intestinal phosphorus absorption data over a wide range of intakes and particularly, those reflecting habitual intakes of individual patients would be ideal. However, we have previously demonstrated that patients with stages 3–4 CKD equilibrate to steady state by 1 week after starting a new controlled dietary intake level.¹⁷ Thus, we believe the effects of any change from habitual intake to our controlled study diet were minimized by the time of the absorption testing on days 7 and 8 of the controlled diet.

The short inpatient study duration of 2 days also is a limitation for interpreting whole-body balance data and particularly, fecal phosphorus data. With shorter balance periods, infrequent fecal samples can lead to artificially high balance (retention) values. Although 2-day balance studies have been successfully reported previously,⁴⁰ we recognize that stool frequency is variable and can be sparse over only 2 days even in the absence of constipation. To help account for this, we normalized the 2-day fecal phosphorus excretion to the expected 2-day excretion of the nonabsorbable fecal marker (PEG). However, the short balance period and infrequent fecal samples are plausible explanations for our balance data showing phosphorus retention in both patients with CKD and healthy controls. Longer-duration balance studies are needed for making more accurate conclusions on absolute values of whole-body phosphorus balance/retention.¹⁸

Strengths of this study included the controlled diet for 6 days prior to the phosphorus absorption testing, the inclusion of two 24-hour uP measurements for a reliable value,¹⁷ inpatient phosphorus absorption testing using a single low-energy radioisotopic tracer to directly assess absorption by kinetic modeling, and the matching of patients to controls by race, sex, and age. There are clinical implications for our findings that intestinal phosphorus absorption in patients with moderate CKD is maintained at levels not detectably different than healthy controls while consuming a phosphorus intake typical in the general United States population, even while 1,25D is reduced. First, phosphorus is absorbed at a relatively high rate in health, and this appears to persist in CKD. Notably, the fractional intestinal phosphorus absorption values observed in our study are similar to those reported by Farrington et al.,¹⁰ who reported data on healthy adults and patients in chronic renal failure, not on dialysis, using a deconvolution analysis of radioisotopic phosphorus, similar to our methods in this study. Farrington et al.¹⁰ reported that cumulative absorption for healthy adults ranged between 60% and 100% at 7.5 hours postradiolabled phosphorus dose, whereas patients with chronic renal failure (mean creatinine clearance of 6±4 ml/min) had a wider range from 20% to 90% at 7.5 hours postradiolabeled phosphorus dose. However, when examining area under the curve for plasma radioactivity, there was no significant difference between groups.¹⁰ Additionally, Scanni et al.⁴¹ used enteral infusion of phosphate to determine absorption efficiency in healthy adults, which they reported as approximately 70%, also consistent with our findings. The high rate of absorption that is maintained in CKD suggests that there is strong rationale for continued efforts to develop better phosphate binders, absorption inhibitors, successful approaches to dietary phosphorus restriction, or other approaches to limit intestinal phosphorus absorption.

In conclusion, our findings indicate that in moderate-stage CKD, intestinal absorption of dietary phosphorus is not detectably reduced compared with healthy adults matched for age, sex, and race while consuming a controlled diet with a high phosphorus intake typical in the United States. This was despite lower 1,25D levels in the patients with CKD. These data underscore the need to develop more effective strategies to address the high intestinal phosphorus absorption in patients with CKD, including strategic dietary phosphorus restriction or modulation to reduce phosphorus burden.

Disclosures

K.M. Hill Gallant has received speaker honorarium from Ardelyx and grant support from Chugai. K.M. Hill Gallant reports ownership interest in the Laboratory for Advanced Medicine (stock). S.M. Moe receives consulting fees from Amgen, Ardelyx, and Sanifit and grant support from Chugai and Keryx/Akebia. S.M. Moe also reports ownership interest in Eli Lilly (stock); and membership on the editorial board of the American Journal of Nephrology. M.E. Wastney is employed by Metabolic Modeling Services. G.N. Wiese reports honoraria from Nutrition Today (Wolters Kluwer). G.N. Wiese is employed by U.S. Renal Care at the time of article publication. E.R. Stremke is employed by Fresenius Medical Care North America at the time of article publication. All remaining authors have nothing to disclose.

Funding

This project was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases http://doi.org/10.13039/100000062 grants K01 DK102864 (to K.M. Hill Gallant), R01 DK110871 (to S.M. Moe), and K23 DK102824 (to R.N. Moorthi); National Institute of Arthritis and Musculoskeletal and Skin Diseases grant P30 AR072581 (to S.M. Moe); and Veterans Administration grant VA Merit BX001471 (to S.M. Moe). The Indiana Clinical and Translational Science Institute is funded in part by National Center for Advancing Translational Sciences award UL1 TR002529. E.R. Stremke received support in part through National Center for Advancing Translational Sciences award TL1 TR002531.