Alex J. Brown

Phosphorus restriction prevents parathyroid gland growth. High phosphorus directly stimulates PTH secretion in vitro.

Dietary phosphorus (P) restriction is known to ameliorate secondary hyperparathyroidism in renal failure patients. In early renal failure, this effect may be mediated by an increase in 1,25-(OH)2D3, whereas in advanced renal failure, P restriction can act independent of changes in 1,25-(OH)2D3 and serum ionized calcium (ICa). In this study, we examined the effects of dietary P on serum PTH, PTH mRNA, and parathyroid gland (PTG) hyperplasia in uremic rats. Normal and uremic rats were maintained on a low (0.2%) or high (0.8%) P diet for 2 mo. PTG weight and serum PTH were similar in both groups of normal rats and in uremic rats fed the 0.2% P diet. In contrast, there were significant increases in serum PTH (130 +/- 25 vs. 35 +/- 3.5 pg/ml, P < 0.01), PTG weight (1.80 +/- 0.13 vs. 0.88 +/- 0.06 microg/gram of body weight, P < 0.01), and PTG DNA (1.63 +/- 0.24 vs. 0.94 +/- 0.07 microg DNA/gland, P < 0.01) in the uremic rats fed the 0.8% P diet as compared with uremic rats fed the 0.2% P diet. Serum ICa and 1,25-(OH)2D3 were not altered over this range of dietary P, suggesting a direct effect of P on PTG function. We tested this possibility in organ cultures of rat PTGs. While PTH secretion was acutely (30 min) regulated by medium calcium, the effects of medium P were not evident until 3 h. During a 6-h incubation, PTH accumulation was significantly greater in the 2.8 mM P medium than in the 0.2 mM P medium (1,706 +/- 215 vs. 1,033 +/- 209 pg/microg DNA, P < 0.02); the medium ICa was 1.25 mM in both conditions. Medium P did not alter PTH mRNA in this system, but cycloheximide (10 microg/ml) abolished the effect of P on PTH secretion. Thus, the effect of P is posttranscriptional, affecting PTH at a translational or posttranslational step. Collectively, these in vivo and in vitro results demonstrate a direct action of P on PTG function that is independent of ICa and 1,25-(OH)2D3.

A new analog of calcitriol, 19-nor-1,25-(OH)2D2, suppresses parathyroid hormone secretion in uremic rats in the absence of hypercalcemia

The active metabolite of vitamin D, calcitriol (1 alpha,25-(OH)2D3), suppresses parathyroid hormone (PTH) gene transcription. Although 1 alpha,25-(OH)2D3 is effective in suppressing secondary hyperparathyroidism (SH) in uremic patients, the mandatory use of large amounts of calcium salts to control serum phosphorus may preclude, in some patients, the use of ideal therapeutic doses of 1 alpha,25-(OH)2D3 because of hypercalcemia. We have studied a new analog of calcitriol, 19-nor-1 alpha,25-(OH)2D2, that possesses low calcemic and phosphatemic activity. Uremic rats received vehicle, 1 alpha,25-(OH)2D3 (2.0, 4.0, or 8.0 ng/rat) or 19-nor-1,25-(OH)2D2 (8.0, 25 or 75 ng/rat) intraperitoneally (IP) every other day for a period of 8 days. Pretreatment and posttreatment values of intact PTH were measured. The normal values for rat intact-PTH were 22 +/- 4.2 pg/mL and for ionized calcium (ICa) 4.77 +/- .07 mg/dL. The only dose of 1 alpha,25-(OH)2D3 that achieved a significantly, suppressed PTH (P < 0.01) was the 8.0 ng/rat. PTH decreased from 202 +/- 31 to 90 +/- 20 pg/mL. However, ICa increased from 4.81 +/- 0.08 to 5.08 mg/dL from uremic control (P < 0.02). Conversely, all doses of 19-nor-1,25-(OH)2D2 were effective in suppressing PTH, and none produced an elevation in ICa that was significantly different from that of vehicle-treated uremic rats. The maximum effect was achieved with the 75 ng/rat dose, which decreased PTH from 193 +/- 49 to 53 +/- 16 pg/mL (a decrease in 72.5%).(ABSTRACT TRUNCATED AT 250 WORDS)

A new analog of 1,25-(OH)2D3, 19-NOR-1,25-(OH)2D2, suppresses serum PTH and parathyroid gland growth in uremic rats without elevation of intestinal vitamin D receptor content

We have previously reported that 19-nor-1,25-(OH)2D2, a new analog of 1,25-(OH)2D3, suppresses parathyroid hormone (PTH) secretion in uremic rats in the absence of hypercalcemia or hyperphosphatemia. In the current study, we examined the effect of 19-nor-1,25-(OH)2D2 on parathyroid gland growth and intestinal vitamin D receptor (VDR) content. After induction of uremia by 5/6 nephrectomy, rats were divided into five experimental groups and received intraperitoneal injections of vehicle, 1,25-(OH)2D3 (2 or 6 ng/rat), or 19-nor-1,25-(OH)2D2 (25 or 100 ng/rat) three times a week for 8 weeks. Twelve normal rats received vehicle and served as the normal control group. During the course of the study, rats were maintained on a 1.0% calcium and 0.8% phosphorus diet. The higher dose of 1,25-(OH)2D3, 6 ng, significantly decreased PTH from 52.7 +/- 10.2 pg/mL in the uremic control group to 25.7 +/- 6.7 pg/mL (P < 0.01). This dose of 1,25-(OH)2D3, however, increased serum levels of both ionized calcium (4.71 +/- 0.05 to 4.85 +/- 0.06 mg/dL; P < 0.05) and phosphorus (4.34 +/- 0.30 to 6.67 +/- 0.63 mg/dL; P < 0.01). Both doses of 19-nor-1,25-(OH)2D2 decreased serum PTH as effectively as 1,25-(OH)2D3 without changes in serum calcium or phosphorus. The 100-ng dose of 19-nor-1,25-(OH)2D2 decreased PTH to 20.7 +/- 3.1 pg/mL (P < 0.01) and suppressed parathyroid gland growth by more than 50%. Both doses of 19-nor-1,25-(OH)2D2 also decreased endogenous 1,25-(OH)2D3 levels compared with uremic control rats (25 ng:30.4 +/- 2.0, P < 0.05, and 100 ng:27.9 +/- 3.2, P < 0.01, v 48.4 +/- 6.6 pg/mL). The 6-ng dose of 1,25-(OH)2D3 elevated intestinal VDR content (138.5 +/- 20.0 fmol/mg protein) compared with animals receiving both doses of 19-nor-1,25-(OH)2D2 (25 ng:84.0 +/- 11.9, P < 0.05, and 100 ng:78.4 +/- 10.9, P < 0.01). This was probably attributable to the marked decrease in endogenous 1,25-(OH)2D3 levels caused by both doses of 19-nor-1,25-(OH)2D2 because intestinal VDR correlated directly with serum 1,25-(OH)2D3 (r = 0.963; P = 0.008). Thus, 19-nor-1,25-(OH)2D2 appears to exert a selective action on the parathyroid glands compared with the intestine. Its low calcemic and phosphatemic properties may result from the decreased endogenous 1,25-(OH)2D3 levels that lead to a reduction in intestinal VDR. This selectivity makes this analog ideal for the treatment of secondary hyperparathyroidism.

Vitamin D analogs: Therapeutic applications and mechanisms for selectivity

The vitamin D endocrine system plays a central role in mineral ion homeostasis through the actions of the vitamin D hormone, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], on the intestine, bone, parathyroid gland, and kidney. The main function of 1,25(OH)(2)D(3) is to promote the dietary absorption of calcium and phosphate, but effects on bone, kidney and the parathyroids fine-tune the mineral levels. In addition to these classical actions, 1,25(OH)(2)D(3) exerts pleiotropic effects in a wide variety of target tissues and cell types, often in an autocrine/paracrine fashion. These biological activities of 1,25(OH)(2)D(3) have suggested a multitude of potential therapeutic applications of the vitamin D hormone for the treatment of hyperproliferative disorders (e.g. cancer and psoriasis), immune dysfunction (autoimmune diseases), and endocrine disorders (e.g. hyperparathyroidism). Unfortunately, the effective therapeutic doses required to treat these disorders can produce substantial hypercalcemia. This limitation of 1,25(OH)(2)D(3) therapy has spurred the development of vitamin D analogs that retain the therapeutically important properties of 1,25(OH)(2)D(3), but with reduced calcemic activity. Analogs with improved therapeutic indices are now available for treatment of psoriasis and secondary hyperparathyroidism in chronic kidney disease, and research on newer analogs for these indications continues. Other analogs are under development and in clinical trials for treatment of various types of cancer, autoimmune disorders, and many other diseases. Although many new analogs show tremendous promise in cell-based models, this article will limit it focus on the development of analogs currently in use and those that have demonstrated efficacy in animal models or in clinical trials.

Successful Treatment of an Adynamic Bone Disorder with Bone Morphogenetic Protein-7 in a Renal Ablation Model

An adynamic bone disorder (ABD) is an important complication of chronic kidney disease (CKD) of unknown etiology for which there is no adequate treatment. Reported is an animal model of ablative CKD complicated by an ABD characterized by the absence of secondary hyperparathyroidism and its successful treatment with a skeletal anabolic factor, bone morphogenetic protein-7 (BMP-7). Adult mice were subjected to electrocautery of the right kidney followed by left nephrectomy. Animals were randomized into groups fed normal chow or fed low-phosphate chow supplemented with calcitriol to maintain normophosphatemia in CKD. All groups were maintained on the regimens for 12 wk. Hyperphosphatemia, secondary hyperparathyroidism, and a mild osteodystrophy developed in the CKD/chow-fed group, as expected. When dietary phosphorus was restricted and calcitriol was administered in the CKD low-phosphate/calcitriol group (ABD), Ca, PO(4), and parathyroid hormone levels were maintained normal. A significant ABD developed in the ABD group characterized by significant depressions in osteoblast number, perimeters, bone formation rates, and mineral apposition rates when compared with the sham-operated, chow-fed group. The abnormal skeletal histomorphometry was reversed by BMP-7 therapy to normal values and significantly improved from the ABD group (P < 0.05). The sham-operated low-phosphate/calcitriol-fed control group and the CKD low-phosphate/calcitriol/BMP-7 groups had reduced phosphate levels compared with the other groups (P < 0.05). ABD produced in mice with CKD in the absence of hyperparathyroidism was successfully reversed with a bone anabolic, BMP-7, associated with a reduction in plasma phosphorus.

1α,25‐Dihydroxy‐3‐Epi‐vitamin D3, a natural metabolite of 1α,25‐dihydroxyvitamin D3, is a potent suppressor of parathyroid hormone secretion

1α,25(OH)2D3 is an important negative regulator of parathyroid hormone (PTH) gene transcription. In parathyroid cells, as in other target tissues, 1α,25(OH)2D3 is degraded by side chain oxidation by the inducible 24‐hydroxylase. We have previously shown that one metabolite of this pathway, 1α,23(S),25‐(OH)3‐24‐oxo‐D3, potently suppresses PTH synthesis and secretion in cultured bovine parathyroid cells (bPTC). Further examination of the metabolites of 1α,25(OH)2D3 in bPTC has revealed another compound that is less polar than 1α,25(OH)2D3. By HPLC analysis and mass spectrometry, this metabolite was identified as 1α,25(OH)2‐3‐epi‐D3. The activity of this metabolite on PTH gene transcription was assessed by the steady‐state PTH secretion by bPTC after 72‐h treatment with concentrations from 10−11 M to 10−7 M. 1α,25(OH)2‐3‐epi‐D3 was found to be only slightly, but not significantly, less active than the native 1α,25(OH)2D3 in suppressing PTH secretion despite having 30 times lower affinity for the bPTC VDR. Both 1α,25(OH)2D3 and 1α,25(OH)2‐3‐epi‐D3 maximally suppressed PTH secretion by 50%. Along with 1α,25(OH)2‐3‐epi‐D3, the activities of the other two A‐ring diastereomers were assessed. 1β,25(OH)2D3 suppressed PTH only at 10−7 M with a decrease of only 30%, in good agreement with its low VDR affinity. Surprisingly, 1β,25(OH)2‐3‐epi‐D3 stimulated PTH secretion by 30–50% at concentrations from 10−11 M to 10− 8M and fell to control (untreated) rates at 10−7M. The mechanism for this increase in PTH secretion is under investigation. Metabolism studies performed in bPTC cells using high concentrations of 1α,25(OH)2D3 substrate showed that in some incubations, the concentration of 1α,25(OH)2‐3‐epi‐D3 was even higher than that of the parent substrate 1α,25(OH)2D3. This finding indicates a slower rate of metabolism for this diastereomer. Thus, production and accumulation of 1α,25(OH)2‐3‐epi‐D3, as a major stable metabolite of 1α,25(OH)2D3 in parathyroid glands, may contribute to the prolonged suppressive effect of 1α,25(OH)2D3 on PTH gene transcription. J. Cell. Biochem. 73:106–113, 1999.

FGF-23 and sFRP-4 in Chronic Kidney Disease and Post Renal Transplantation

Background: The phosphatonins fibroblast growth factor-23 (FGF-23) and FRP-4 are inhibitors of tubular phosphate reabsorption that may play a role in the hyperphosphatemia associated with chronic kidney disease (CKD) or in the hypophosphatemia associated with renal transplants. Methods: Plasma FGF-23, FRP-4, phosphorus and parathyroid hormone were measured in patients at all stages of CKD. Phosphate regulation of FGF-23 and secreted frizzled related protein-4 (sFRP-4) was examined in end-stage renal disease patients in the presence and absence of therapeutic phosphate binder usage. In renal transplant patients, plasma FGF-23, sFRP-4 and phosphorus concentrations were determined before and 4–5 days after transplantation. Results: Plasma FGF-23 correlated with creatinine clearance (r2 = –0.584, p < 0.0001) and plasma phosphorus (r2 = 0.347, p < 0.001) in CKD patients and with plasma phosphorus (r2 = 0.448, p < 0.001) in end-stage renal disease patients. Phosphate binder withdrawal increased FGF-23 levels. In kidney transplant patients, dramatic decreases in FGF-23 (–88.8 ± 5.4%) and phosphorus (–64 ± 10.2%) were observed by 4–5 days post-transplantation. In patients with post-transplant hypophosphatemia, FGF-23 levels correlated inversely with plasma phosphorus (r2 = 0.661, p < 0.05). sFRP-4 levels did not change with creatinine clearance or hyperphosphatemia in CKD or end-stage renal disease patients, and no relation was noted between post-transplant sFRP-4 levels and hypophosphatemia. Conclusions: In CKD, FGF-23 levels rose with decreasing creatinine clearance rates and increasing plasma phosphorus levels, and rapidly decreased post-transplantation suggesting FGF-23 is cleared by the kidney. Residual FGF-23 may contribute to the hypophosphatemia in post-transplant patients.

The Role of Phosphorus in the Development of Secondary Hyperparathyroidism and Parathyroid Cell Proliferation in Chronic Renal Failure

Hyperplasia of the parathyroid glands and high levels of parathyroid hormone (PTH) are among the most consistent findings in patients with chronic renal failure. In early renal failure, alterations in vitamin D metabolism play a key role in the development of secondary hyperparathyroidism. Low levels of calcitriol and decreased expression of the vitamin D responsive element may allow greater synthesis and secretion of PTH. Phosphorus independent of serum calcium and calcitriol increases PTH synthesis and secretion by a post-transcriptional mechanism. Studies in vivo in uremic rats demonstrated that an increase in dietary phosphorus induces parathyroid gland hyperplasia. If the rats are then fed a low-phosphorus diet, the levels of serum PTH return to normal; however, the size of the parathyroid glands remains enlarged. No apoptosis was observed in the glands. To further characterize the effects of phosphorus on PTH synthesis and secretion, intact rat parathyroid glands were metabolically labeled during a 4-hour incubation in methionine-free medium containing 1.25 mM Ca2+, [35S]methionine, and either 2.8 mM or 0.2 mM phosphorus. Total PTH secretion, as measured in the medium, was increased more than 6-fold in glands incubated in high-phosphorus medium compared with glands incubated in the low-phosphorus medium. Thus, in the past 20 years, numerous investigators have provided strong evidence for the action of phosphorus on PTH secretion. Unfortunately, the absence of a parathyroid cell line is slowing the progress in understanding the molecular mechanism(s) involved in phosphorus regulation of PTH.

Drug Insight: vitamin D analogs in the treatment of secondary hyperparathyroidism in patients with chronic kidney disease

Secondary hyperparathyroidism commonly develops in patients with chronic kidney disease (CKD) in response to high phosphate, low calcium and low 1α,25-dihydroxyvitamin D3 (calcitriol) levels. High levels of parathyroid hormone (PTH) accelerate bone turnover, with efflux of calcium and phosphate that can lead to vascular calcification. Treatment of secondary hyperparathyroidism with calcitriol and calcium-based phosphate binders can produce hypercalcemia and oversuppression of PTH, which results in adynamic bone that cannot buffer calcium and phosphate levels, and increased risk of vascular calcification. PTH levels must, therefore, be reduced to within a range that supports normal bone turnover and minimizes ectopic calcification. Vitamin D analogs that inhibit PTH gene transcription and parathyroid hyperplasia (and have reduced calcemic activity) are a safer treatment for secondary hyperparathyroidism than calcitriol; these agents enhance the survival of patients with CKD. Several such analogs are now in use, and analogs with even greater selectivity than those currently used are in development. Parathyroid glands express both 25-hydroxylase and 1α-hydroxylase, which suggests that these enzymes might suppress parathyroid function by an autocrine mechanism. The risk of hypercalcemia with vitamin D analog therapy is reduced by the introduction of non-calcium-based phosphate binders and cinacalcet; furthermore, recent trials indicate that early intervention with vitamin D analogs in stage 3 and 4 CKD can correct PTH levels, and could prevent renal bone disease and prolong patient survival.

Reversal of Secondary Hyperparathyroidism by Phosphate Restriction Restores Parathyroid Calcium-Sensing Receptor Expression and Function

Secondary hyperparathyroidism (2° HPT), a common disorder in chronic renal failure (CRF) patients, is characterized by hypersecretion of parathyroid hormone (PTH), parathyroid hyperplasia, and decreased expression of the calcium‐sensing receptor (CaR). Dietary phosphate loading promotes 2° HPT, and phosphate restriction prevents and arrests 2° HPT in CRF. This study examined the ability of phosphate restriction to restore parathyroid CaR expression and function. Uremic rats fed a 1.2% P diet for 2 weeks developed 2° HPT with down‐regulated CaR expression. Continuation on the 1.2% P diet for 2 more weeks worsened the 2° HPT and further decreased CaR, but switching the rats to a 0.2% P diet for 2 weeks normalized PTH, arrested parathyroid hyperplasia, and restored CaR expression to normal. The calcium‐PTH relationship was abnormal in uremic rats fed a high phosphate (HP) diet with a right‐shifted calcium set point but was corrected by 2 weeks of phosphate restriction. A time course revealed that following the switch to a low phosphate diet, PTH levels were normalized by day 1, and growth was arrested by day 2, but CaR expression was restored between days 7 and 14. We conclude that although phosphate restriction restores CaR expression and function in parathyroid glands of uremic rats, it is a late event and not involved in the arrest of 2° HPT.