Anthony A. Portale

Effect of dietary phosphorus on circulating concentrations of 1,25-dihydroxyvitamin D and immunoreactive parathyroid hormone in children with moderate renal insufficiency.

The hyperparathyroidism characteristic of patients with moderate renal insufficiency could be caused by decreases in the plasma concentration of ionized calcium (Ca++) evoked by: (a) recurring increases in the plasma concentration of inorganic phosphorus that may be detectable only in the post-prandial period; (b) a reversible, phosphorus-mediated suppression of renal 25-hydroxyvitamin D-1 alpha-hydroxylase that decreases the plasma concentration of 1,25-dihydroxyvitamin D (1,25-(OH)2D) enough to decrease both gut absorption and bone resorption of Ca++; (c) both of these. In a group of eight children with moderate renal insufficiency, mean glomerular filtration rate (GFR) 45 +/- 4 (SE) ml/min per 1.73 M2, ages 6-17 yr, we tested these hypotheses by determining the effect of short term (5 d) restriction and supplementation of dietary intake of phosphorus on the plasma concentration of 1,25-(OH)2D, the serum concentrations of immunoreactive parathyroid hormone (iPTH) and phosphorus, and the fractional renal excretion of phosphorus ( FEPi ). When dietary phosphorus was normal, 1.2 g/d, the serum concentrations of phosphorus throughout the day were not greater than those of normal control children, and the serum concentrations of carboxyl-terminal iPTH (C-iPTH) were greater, 59 +/- 9 vs. 17 +/- 3 mu leq/ml, and unchanging; the serum concentration of intact-iPTH was also greater, 198 +/- 14 vs. 119 +/- 8 pg/ml. The plasma concentration of 1,25-(OH)2D was lower than that of age-matched controls, 27 +/- 3 vs. 36 +/- 2 pg/ml (P less than 0.01). When dietary phosphorus was restricted to 0.35 g/d, the plasma concentration of 1,25-(OH)2D increased by 60% to a mean value not different from that of normal controls, while serum concentrations of C-iPTH and intact-iPTH decreased by 25%, the latter concentration to a mean value not different from that of controls. FEPi decreased from 31 to 9%. When dietary phosphorus was supplemented to 2.4 g/d, the plasma concentration of 1,25-(OH)2D decreased 32%, while those of C-iPTH and intact-iPTH increased by 131 and 45%, respectively; FEPi increased from 27 to 53%. Plasma concentrations of 25-hydroxyvitamin D remained normal and unchanged, and GFR did not change when dietary phosphorus was manipulated. The data demonstrate that in children with moderate renal insufficiency: (a) A normal dietary intake of phosphorus in attended by a decreased circulating concentration of 1,25-(OH)2D and an increased concentration of iPTH, but not by recurring increases in the serum concentration of phosphorus at any time of the day; (b) Dietary phosphorus is, however, a major determinant of the circulating concentrations of both 1,25-(OH)2D and iPTH, which vary inversely and directly, respectively, with dietary intake of phosphorus, and increase and decrease, respectively, to normal values when phosphorus is restricted for 5 d; (c) Restriction and supplementation of dietary phosphorus induces changes in the serum concentration of iPTH that correlate strongly but inversely with those induced in the plasma concentration of 1,25-(OH)2D (r = -0.88, P < 0.001); and (d) The physiologic responsiveness of the renal tubule to changes in dietary phosphorus is to a substantial extent intact. The data provide support for the second hypothesis stated.

Oral intake of phosphorus can determine the serum concentration of 1,25-dihydroxyvitamin D by determining its production rate in humans.

Changes in the oral intake of phosphorus could induce the reported changes in the serum concentration of 1,25-dihydroxyvitamin D (1,25-(OH)2D) by inducing changes in its production rate (PR) or metabolic clearance rate (MCR), or both. To investigate these possibilities, we employed the constant infusion equilibrium technique to measure the PR and MCR of 1,25-(OH)2D in six healthy men in whom the oral intake of phosphorus was initially maintained at 1,500 mg/70 kg body weight per d for 9 d, then restricted to 500 mg/d (coupled with oral administration of aluminum hydroxide) for 10 d, and then supplemented to 3,000 mg/d for 10 d. With phosphorus restriction, the serum concentration of 1,25-(OH)2D increased by 80% from a mean of 38 +/- 3 to 68 +/- 6 pg/ml, P less than 0.001; the PR increased from 1.8 +/- 0.2 to 3.8 +/- 0.6 micrograms/d, P less than 0.005; the MCR did not change significantly. The fasting serum concentration of phosphorus decreased from 3.5 +/- 0.2 to 2.6 +/- 0.2 mg/dl, P less than 0.01. With phosphorus supplementation, the serum concentration of 1,25-(OH)2D decreased abruptly, reaching a nadir within 2 to 4 d; after 10 d of supplementation, the mean concentration of 27 +/- 4 pg/ml was lower by 29%, P less than 0.01, than the value measured when phosphorus intake was normal. The PR decreased to 1.3 +/- 0.2 micrograms/d, P less than 0.05; the MCR did not change significantly. The fasting serum concentration of phosphorus increased significantly, but only initially. These data demonstrate that in healthy men, reductions and increases in the oral intake of phosphorus can induce rapidly occurring, large, inverse, and persisting changes in the serum concentration of 1,25-(OH)2D. Changes in the PR of 1,25-(OH)2D account entirely for the phosphorus-induced changes in serum concentration of this hormone.

Physiologic regulation of the serum concentration of 1,25-dihydroxyvitamin D by phosphorus in normal men.

We asked this question: in normal humans, is either a normal dietary intake or normal serum concentration of phosphorus a determinant of the serum concentration of 1,25(OH)2D? In seven normal men whose dietary phosphorus was decreased from 2,300 to 625 mg/d, each intake for 8-9 d, under strictly controlled, normal metabolic conditions, we measured serum concentrations of 1,25(OH)2D daily, and concentrations of phosphorus hourly throughout a 24-h period, before and after restriction. Decreasing dietary phosphorus induced: (a) a 58% increase in serum levels of 1,25(OH)2D; (b) a 35% decrease in serum levels of phosphorus measured in the afternoon; (c) a 12% decrease in the 24-h mean serum level of phosphorus; but, (d) no decrease in morning fasting levels of phosphorus. Serum concentrations of 1,25(OH)2D varied inversely and significantly with 24-h mean concentrations of phosphorus (r = -0.77, P less than 0.001). When these data are combined with those of our prior study in which dietary phosphorus was varied over an extreme range, the relationship between serum levels of 1,25(OH)2D and 24-h mean serum levels of phosphorus is even stronger (r = -0.90, P less than 0.001). In the aggregate, the results demonstrate that in normal men, dietary phosphorus throughout a normal range and beyond, can finely regulate the renal production and serum concentration of 1,25(OH)2D, and provide evidence that this regulation is mediated by fine modulation of the serum concentration of phosphorus.

Dietary intake of phosphorus modulates the circadian rhythm in serum concentration of phosphorus. Implications for the renal production of 1,25-dihydroxyvitamin D.

We recently reported that in healthy men, changes in the production rate (PR) of 1,25-dihydroxyvitamin D [1,25-(OH)2D] accounted for the 80% increase and the 30% decrease in its serum concentration that was induced by restriction and supplementation, respectively, of dietary phosphorus. These changes in PR and serum concentration of 1,25-(OH)2D could be mediated by changes in serum concentrations of phosphorus that occur after the morning fasting period. To examine this hypothesis, we measured serum concentrations of phosphorus in blood drawn at hourly intervals for 24 h in six healthy men in whom dietary phosphorus was initially maintained at 1,500 mg/70 kg body weight per day for 9 d, then restricted to 500 mg/d (coupled with orally administered aluminum hydroxide) for 10 d, and then supplemented to 3,000 mg/d for 10 d. When dietary phosphorus was normal, the serum concentration of phosphorus exhibited the normal circadian rhythm: a rapid decrease in early morning to a nadir at 1100, followed by an increase to plateau at 1600 h and a further increase to an acrophase (peak) at 0030 h. The variation in serum levels of phosphorus can be described as the sum of sinusoidal functions with periodicities of 24 and 12 h. Phosphorus restriction for 10 d induced a 40% reduction in the 24-h mean serum level of phosphorus, abolished the early afternoon rise in its serum level (i.e., the 12-h periodic component of the time series), and delayed the acrophase by 3 h to 0330 h. Phosphorus supplementation for 10 d induced a 14% increase in the 24-h mean serum level of phosphorus but no significant change in its morning fasting level, exaggerated the early afternoon rise in serum phosphorus, and advanced the acrophase by 9 h to 1530 h. The changes in the PR of 1,25-(OH)2D induced by restriction and supplementation of dietary phosphorus varied inversely and significantly with those induced in the 24-h mean serum level of phosphorus (R = -0.88, P less than 0.001). These data demonstrate that in healthy men, dietary phosphorus is an important determinant of the serum concentration of phosphorus throughout most of the day. The data suggest that diet-induced changes in serum levels of phosphorus mediate the changes in PR and serum concentration of 1,25(OH)2D.

A Peripheral Blood Diagnostic Test for Acute Rejection in Renal Transplantation

Monitoring of renal graft status through peripheral blood (PB) rather than invasive biopsy is important as it will lessen the risk of infection and other stresses, while reducing the costs of rejection diagnosis. Blood gene biomarker panels were discovered by microarrays at a single center and subsequently validated and cross‐validated by QPCR in the NIH SNSO1 randomized study from 12 US pediatric transplant programs. A total of 367 unique human PB samples, each paired with a graft biopsy for centralized, blinded phenotype classification, were analyzed (115 acute rejection (AR), 180 stable and 72 other causes of graft injury). Of the differentially expressed genes by microarray, Q‐PCR analysis of a five gene‐set (DUSP1, PBEF1, PSEN1, MAPK9 and NKTR) classified AR with high accuracy. A logistic regression model was built on independent training‐set (n = 47) and validated on independent test‐set (n = 198)samples, discriminating AR from STA with 91% sensitivity and 94% specificity and AR from all other non‐AR phenotypes with 91% sensitivity and 90% specificity. The 5‐gene set can diagnose AR potentially avoiding the need for invasive renal biopsy. These data support the conduct of a prospective study to validate the clinical predictive utility of this diagnostic tool.

Dietary Phosphorus Transcriptionally Regulates 25-Hydroxyvitamin D-1α-Hydroxylase Gene Expression in the Proximal Renal Tubule

Synthesis of the hormone 1,25-dihydroxyvitamin D, the biologically active form of vitamin D, occurs in the kidney and is catalyzed by the mitochondrial cytochrome P450 enzyme, 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase). We sought to characterize the effects of changes in dietary phosphorus on the kinetics of renal mitochondrial 1alpha-hydroxylase activity and the renal expression of P450c1alpha and P450c24 mRNA, to localize the nephron segments involved in such regulation, and to determine whether transcriptional mechanisms are involved. In intact mice, restriction of dietary phosphorus induced rapid, sustained, approximately 6- to 8-fold increases in renal mitochondrial 1alpha-hydroxylase activity and renal P450c1alpha mRNA abundance. Immunohistochemical analysis of renal sections from mice fed the control diet revealed the expression of 1alpha-hydroxylase protein in the proximal convoluted and straight tubules, epithelial cells of Bowmans capsule, thick ascending limb of Henles loop, distal tubule, and collecting duct. In mice fed a phosphorus-restricted diet, immunoreactivity was significantly increased in the proximal convoluted and proximal straight tubules and epithelial cells of Bowmans capsule, but not in the distal nephron. Dietary phosphorus restriction induced a 2-fold increase in P450c1alpha gene transcription, as shown by nuclear run-on assays. Thus, the increase in renal synthesis of 1,25-dihydroxyvitamin D induced in normal mice by restricting dietary phosphorus can be attributed to an increase in the renal abundance of P450c1alpha mRNA and protein. The increase in P450c1alpha gene expression, which occurs exclusively in the proximal renal tubule, is due at least in part to increased transcription of the P450c1alpha gene.

Being Overweight Modifies the Association Between Cardiovascular Risk Factors and Microalbuminuria in Adolescents

OBJECTIVE. The goal was to determine the association between cardiovascular risk factors and microalbuminuria in a nationally representative sample of adolescents and to determine whether being overweight modifies this association. METHODS. We analyzed cross-sectional data from the National Health and Nutrition Examination Survey(1999–2004) for 2515 adolescents 12 to 19 years of age. Cardiovascular risk factors included abdominal obesity, impaired fasting glucose, diabetes mellitus, insulin resistance, high triglyceride levels, low high-density lipoprotein cholesterol levels, hypertension, smoking, and the metabolic syndrome. Microalbuminuria was defined as a urinary albumin/creatinine ratio of 30 to 299 mg/g in a random morning sample. Overweight was defined as BMI of ≥95th percentile, according to the Centers for Disease Control and Prevention 2000 growth charts. RESULTS. Microalbuminuria was present in 8.9% of adolescents. The prevalence of microalbuminuria was higher among nonoverweight adolescents than among overweight adolescents. The median albumin/creatinine ratio decreased with increasing BMI z scores. The association of microalbuminuria with cardiovascular risk factors differed according to BMI category. Among nonoverweight adolescents, microalbuminuria was not associated with any cardiovascular disease risk factor except for overt diabetes mellitus. Among overweight adolescents, however, microalbuminuria was associated with impaired fasting glucose, insulin resistance, hypertension, and smoking, as well as diabetes mellitus. CONCLUSION. For the majority of adolescents, microalbuminuria is not associated with cardiovascular risk factors. Among overweight adolescents, however, microalbuminuria is associated with cardiovascular risk factors. The prognostic importance of microalbuminuria in overweight and nonoverweight adolescents with regard to future cardiovascular and renal disease needs to be defined in prospective studies conducted specifically in children.

Vitamin D 1α-Hydroxylase

The rate-limiting, hormonally regulated step in the bioactivation of vitamin D is renal 1 alpha-hydroxylation by P450c1 alpha. In late 1997, we reported the cloning of the human cDNA and gene from keratinocytes, and established that P450c1 alpha mutations cause vitamin D-dependent rickets, type I, while three other groups reported the cloning of the rodent enzyme. The genetics of P450c1 alpha are well established, with studies of over 30 patients, but the molecular mechanisms for the hormonal regulation of P450c1 alpha are still under investigation.

Complete Steroid Avoidance Is Effective and Safe in Children With Renal Transplants: A Multicenter Randomized Trial With Three‐Year Follow‐Up

To determine whether steroid avoidance in pediatric kidney transplantation is safe and efficacious, a randomized, multicenter trial was performed in 12 pediatric kidney transplant centers. One hundred thirty children receiving primary kidney transplants were randomized to steroid‐free (SF) or steroid‐based (SB) immunosuppression, with concomitant tacrolimus, mycophenolate and standard dose daclizumab (SB group) or extended dose daclizumab (SF group). Follow‐up was 3 years posttransplant. Standardized height Z‐score change after 3 years follow‐up was –0.99 ± 2.20 in SF versus –0.93 ± 1.11 in SB; p = 0.825. In subgroup analysis, recipients under 5 years of age showed improved linear growth with SF compared to SB treatment (change in standardized height Z‐score at 3 years –0.43 ± 1.15 vs. –1.07 ± 1.14; p = 0.019). There were no differences in the rates of biopsy‐proven acute rejection at 3 years after transplantation (16.7% in SF vs. 17.1% in SB; p = 0.94). Patient survival was 100% in both arms; graft survival was 95% in the SF and 90% in the SB arms (p = 0.30) at 3 years follow‐up. Over the 3 year follow‐up period, the SF group showed lower systolic BP (p = 0.017) and lower cholesterol levels (p = 0.034). In conclusion, complete steroid avoidance is safe and effective in unsensitized children receiving primary kidney transplants.

The Mechanism of 1,25-Dihydroxyvitamin D3Autoregulation in Keratinocytes

The synthesis of 1,25-dihydroxyvitamin D3(1,25(OH)2D3) from its precursor, 25-dihydroxyvitamin D3 (25(OH)D3), is catalyzed by the mitochondrial cytochrome P450 enzyme 25-hydroxyvitamin D3-1α-hydroxylase (1α-hydroxylase). It has been generally assumed that 1,25(OH)2D3inhibits the activity of this enzyme by regulating its expression at the genomic level. We confirmed that 1,25(OH)2D3 reduced the apparent conversion of 25(OH)D3 to 1,25(OH)2D3 while stimulating the conversion of 1,25(OH)2D3 and 25(OH)D3 to 1,24,25(OH)3D3 and 24,25(OH)2D3, respectively. However, 1,25(OH)2D3 failed to reduce the abundance of its mRNA or its encoded protein in human keratinocytes. Instead, when catabolism of 1,25(OH)2D3 was blocked with a specific inhibitor of the 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) all apparent inhibition of 1α-hydroxylase activity by 1,25(OH)2D3 was reversed. Thus, the apparent reduction in 1α-hydroxylase activity induced by 1,25(OH)2D3 is due to increased catabolism of both substrate and product by the 24-hydroxylase. We believe this to be a unique mechanism for autoregulation of steroid hormone synthesis.