Granulocitos de baja densidad: Un nuevo marcador de deterioro óseo en pacientes en diálisis peritoneal / Low-density granulocytes: A new marker of bone deterioration in peritoneal dialysis patients

Objetivo: En enfermos renales, la enfermedad ósea-metabólica, la inflamación sistémica y la malnutrición exacerban el riesgo de calcificación vascular (CV) y la morbimortalidad. Dada la fuerte asociación entre CV y fracturas por fragilidad, el objetivo de este estudio es evaluar la contribución de los mayores determinantes de CV al deterioro óseo en pacientes en diálisis peritoneal (DP).Métodos: En 31 pacientes no diabéticos en DP (>6 meses), se estudiaron marcadores de alteraciones del metabolismo óseo, daño vascular, inflamación y desnutrición, y, su impacto en el deterioro óseo (osteopenia radiológica y/o antecedentes de fractura por fragilidad).Resultados: En estos pacientes, (20 varones y 11 mujeres; edad=54±15 y 60±11 años respectivamente (p=0,24)), la prevalencia de fracturas por fragilidad fue de 5% en hombres y del 27% en mujeres. El deterioro óseo fue mayor en personas de edad avanzada, sexo femenino, índices de Charlson y Kauppila elevados, menor masa muscular y con expansión de una subpoblación altamente inflamatoria de granulocitos inmaduros de baja densidad (LDGi). Un análisis de regresión logística demostró que el riesgo de deterioro óseo está más influenciado por el sexo femenino que por la edad y que, de los múltiples factores asociados a mayor deterioro óseo estudiados, sólo la expansión de LDGi estima el riesgo de alteraciones óseas en estos pacientes independientemente de su edad y sexo.Conclusión: La expansión de LDGi provee de un biomarcador certero para el diagnóstico de deterioro óseo y para monitorizar estrategias que atenúen su progresión en pacientes en DP de cualquier edad y sexo. (AU)

Hipometilación del gen de la PTH por elevado fósforo de la dieta: un posible agravante epigenético de la severidad del hiperparatiroidismo secundario en la enfermedad renal crónica / Hypomethylation of the PTH gene due to high dietary phosphorus: a possible aggravating of severe secondary hyperparathyroidism in chronic renal failure

Introducción: En pacientes con enfermedad renal crónica (ERC), la hiperfosfatemia agrava tanto la hiperplasia paratiroidea como la síntesis y secreción de PTH. La mayor hiperplasia se asocia a descensos en la expresión génica de los receptores de calcio (CaSR), vitamina D (VDR) y también de α-Klotho, induciendo resistencia de la glándula paratiroides para responder tanto al tratamiento como a los aumentos de FGF23. Este estudio examinó la posible contribución epigenética del fósforo elevado en agravar el hiperparatiroidismo secundario (HPTS). Material y métodos: Se comparó el grado de metilación mediante pirosecuenciación de bisulfito en secuencias ricas en CpG de los promotores en los genes del CaSR, VDR, PTH y α-Klotho en ADN de glándulas paratiroides de ratas urémicas alimentadas con dieta con contenido normal y elevado en fósforo. Resultados: La dieta rica en fósforo incrementó la expresión de PTH y causó una marcada reducción del grado de metilación en el promotor del gen de PTH. En cambio, las regiones promotoras de los genes de CaSR, VDR y α-Klotho no mostraron diferencias significativas en el porcentaje de metilación entre ambos grupos de ratas, no siendo, por tanto, éste el mecanismo determinante de la disminución de la expresión de estos genes observada en el HPTS. Conclusiones: Las alteraciones epigenéticas inducidas por la dieta rica en fósforo en el HPTS, en particular la hipometilación del gen de la PTH, podrían contribuir a los aumentos que se producen en la síntesis y secreción de esta hormona. La identificación de los mecanismos implicados permitiría diseñar mejores tratamientos para el HPTS en fases tempranas de la ERC (AU)

Introduction: Hyperphosphataemia aggravates both parathyroid hyperplasia and PTH secretion in patients with chronic kidney disease (CKD). Hyperplasia is associated with decreases in calcium receptor expression (CaSR), vitamin D (VDR) and α-Klotho, inducing resistance of the parathyroid gland to respond both to treatment and to increases in FGF23. This study examined the possible epigenetic contributions of raised phosphorus to aggravate secondary hyperparathyroidism (SHPT) in patients with (CRD). Material and methods: The degree of methylation was compared by pyrosequencing of bisulfite in CpGrich sequences of the promoters in the CaSR, VDR, PTH and α-Klotho genes in parathyroid gland DNA from uremic rats fed a normal and high phosphorus diet. Results: The diet rich in phosphorus increased PTH expression and caused a marked reduction in the degree of methylation in the promoter of the PTH gene. In contrast, the promoter regions of the CaSR, VDR and α-Klotho genes did not show significant differences in the percentage of methylation between the two groups of rats. Thus, it was not the determining mechanism for the decrease of the expression of these genes observed in the SHPT. Conclusions: The epigenetic alterations induced by the phosphorus rich diet in SHPT, particularly the PTH gene hypomethylation, could contribute to the increases that occur in the synthesis and secretion of this hormone. The identification of the mechanisms involved would allow better treatments for SHPT to be designed in the early stages of CKD (AU)

Pathogenic mechanisms for parathyroid hyperplasia.

Parathyroid hyperplasia is the cause of parathyroid gland enlargement in kidney disease (KD). Hypocalcemia, hyperphosphatemia, and vitamin D deficiency are critical contributors to the worsening of the hyperplastic parathyroid growth induced by KD. Reproduction of the features of human KD in the 5/6 nephrectomized rat model has shown that 80% of the mitogenic signals induced by KD in parathyroid cells that are aggravated by either high phosphate (P) or low calcium (Ca) diets occurred within 5 days after the onset of KD. Enhanced parathyroid expression of the potent growth promoter transforming growth factor alpha (TGFalpha) and its receptor, the epidermal growth factor receptor (EGFR), was identified as the main cause of parathyroid hyperplasia in experimental KD. Indeed, administration of highly specific EGFR-tyrosine kinase inhibitors (TKI), which block downstream signaling from TGFalpha-activated EGFR, completely prevented high P- and low Ca-induced parathyroid hyperplasia in early KD, as well as the severe progression of high P-induced parathyroid growth in established secondary hyperparathyroidism, the latter characterized by marked TGFalpha and EGFR overexpression in the parathyroid glands. More importantly, the suppression of signals downstream from TGFalpha binding to EGFR with EGFR-TKI treatment also revealed that TGFalpha self-upregulation in the parathyroid glands is the main determinant of the severity of the hyperplastic growth, and that enhanced TGFalpha activation of EGFR mediates the reduction in parathyroid vitamin D receptor levels thereby causing resistance to both the antiproliferative and parathyroid hormone-suppressive properties of calcitriol therapy.

1alpha-Hydroxylase transactivation by gamma-interferon in murine macrophages requires enhanced C/EBPbeta expression and activation.

gamma-Interferon [gamma-IFN] induction of macrophage 1alpha-hydroxylase mRNA and activity causes severe hypercalcemia in granulomatous disorders. These studies demonstrate transcriptional regulation. gamma-IFN induces the activity of the murine 1alpha-hydroxylase [-1651; +22] promoter in the murine macrophage cell line Raw 264.7 only after a 24h exposure. This slow kinetics is incompatible with classical gamma-IFN-mediated transactivation. In fact, gamma-IFN response mapped to the minimal [-85; +11] promoter, which lacks GAS or ISRE sites but contains a putative C/EBPbeta site. C/EBPbeta is a gamma-IFN inducible gene and a novel mediator of gamma-IFN-regulated transcription. As expected for a C/EBPbeta-driven transcription, ectopic C/EBPbeta expression was sufficient to increase 1alpha-hydroxylase activity, enhance minimal promoter activity and potentiate the induction of this promoter by gamma-IFN. Importantly, the dominant negative C/EBPbeta isoform antagonized C/EBPbeta-transcriptional activity. gamma-IFN induction of C/EBPbeta expression is not sufficient for gamma-IFN induction of minimal promoter activity. There is also a cell-specific induction of C/EBPbeta-transcriptional activity by gamma-IFN. In Raw cells, specific inhibition of gamma-IFN induction of endogenous-C/EBPbeta phosphorylation by MEKK1 markedly reduced basal promoter activity and the response to gamma-IFN. We conclude that gamma-IFN-induction of C/EBPbeta expression and activation by phosphorylation contributes to gamma-IFN-transcriptional control of 1alpha-hydroxylase expression in murine macrophages.

p21WAF1 and TGF-alpha mediate parathyroid growth arrest by vitamin D and high calcium.

BACKGROUND: High dietary phosphorus (P) worsens uremia-induced parathyroid (PT) hyperplasia through increases in the growth promoter transforming growth factor-alpha (TGF-alpha). In contrast, P restriction prevents PT hyperplasia by inducing the cell cycle inhibitor p21. Since 1,25(OH)2D3-antiproliferative action in various cell types involve increases in p21, we studied whether induction of p21 by 1,25(OH)2D3 or the vitamin D analog, 19-Nor-1,25(OH)2D2, could counteract the PT hyperplasia induced by high dietary P in early uremia. METHODS: Normal (N) and uremic (U; 5/6 nephrectomized) female Sprague-Dawley rats were fed high P (HP), low P (LP) or high Ca (HCa) diets and administered intraperitoneally (IP) either vehicle or vitamin D metabolites for seven days, as follows: N-HP; U-HP + vehicle; U-HP + 1,25(OH)2D3 (4 ng/day); U-HP + 19-Nor-1,25(OH)2D2 (30 ng/day); U-LP; U-HCa. Serum PTH and PT gland weight assessed secondary hyperparathyroidism. Immunohistochemical quantitation of two markers of mitotic activity, Ki67 and PCNA measured PT hyperplasia. Immunohistochemical expression of PT p21 and TGF-alpha addressed potential mechanisms regulating PT cell growth. RESULTS: 1,25(OH)2D3 and 19-Nor-1,25(OH)2D2 were effective in suppressing both PTH secretion and PT hyperplasia induced by uremia and high dietary P independent of increases in ionized Ca. Both vitamin D compounds enhanced PT p21 expression and prevented high P-induced increases in PT TGF-alpha content. Induction of PT p21 and reduction of TGF-alpha content also occurred when uremia-induced PT hyperplasia was suppressed by high dietary Ca. CONCLUSIONS: In early uremia, vitamin D suppression of high P-induced PT hyperplasia and high dietary Ca arrest of PT growth involve induction of PT p21 and prevention of increases in TGF-alpha.

p21(WAF1) and transforming growth factor-alpha mediate dietary phosphate regulation of parathyroid cell growth.

BACKGROUND: The parathyroid (PT) hyperplasia induced by renal failure can be further enhanced by high dietary phosphate (P) or completely abolished by P restriction. To identify potential mechanisms mediating these opposing effects of dietary P on PT growth, this study first focused on p21(WAF1) (p21) because high P reduces while low P enhances serum 1,25-dihydroxyvitamin D, whose potent antiproliferative properties result from the induction of p21. In addition to reducing p21, high P-induced PT growth could result from increased PT expression of the growth promoter transforming growth factor-alpha (TGF-alpha), known to be elevated in hyperplastic and adenomatous human PT glands. METHODS: The time course for dietary P regulation of PT expression of TGF-alpha and p21 was assessed for seven days after 5/6 nephrectomy in rats and correlated with the degree of PT hyperplasia and secondary hyperparathyroidism. RESULTS: In P-restricted 5/6 nephrectomized rats, PT-p21 mRNA and protein increased by day 2, independent of changes in serum 1,25-dihydroxyvitamin D, and remained higher than in the high P counterparts for up to seven days. The PT hyperplasia of the high P group could not be attributed to a reduction of PT-p21 expression from normal control values. Instead, PT-TGF-alpha protein was higher in uremic rats compared with normal controls and increased further with high dietary P intake. PT levels of proliferating cell nuclear antigen (PCNA), an index of cell mitoses, correlated inversely with p21 and directly with TGF-alpha. Consistent with these findings, PT gland size and serum PT hormone levels, similar in both dietary groups at day 2, were higher in the high P group by day 5. Induction of p21 by low P and of TGF-alpha by high P was specific for the PT glands. Dietary P had no effect either on intestinal growth or p21 or TGF-alpha protein content. CONCLUSIONS: These findings suggest that low P induction of p21 could prevent PT hyperplasia in early uremia, whereas high P enhancement of TGF-alpha may function as an autocrine signal to stimulate growth further.

Role of phosphorus in the pathogenesis of secondary hyperparathyroidism.

Secondary hyperparathyroidism (SH) and hyperplasia of the parathyroid glands (PTG) are universal complications in patients with CRF. In early renal failure, reduction in serum calcitriol and moderate decreases in ionized calcium contribute to greater synthesis and secretion of PTH. As renal disease progresses, a reduction in parathyroid expression of vitamin D receptor and calcium receptor renders the PTG more resistant to both calcitriol and calcium. High dietary phosphorus (P), independent of calcium and calcitriol, further enhances uremia-induced PTG hyperplasia and PTH synthesis and secretion, the latter by posttranscriptional mechanisms. Once SH develops, dietary P restriction can return the high serum PTH levels toward normal, however, parathyroid hyperplasia persists. Studies in our laboratory identified 2 of the mechanisms involved in the opposing effects of high and low dietary P content on PTG growth. Whereas high dietary P increases parathyroid expression of transforming growth factor alpha (TGFalpha), a growth promoter, P restriction induces the cyclin-dependent kinase inhibitor p21, an inducer of growth arrest. Both effects of P are specific for the PTG. No increase in either protein was observed in liver or intestine. TGFalpha induction of hyperplasia involves binding to the epidermal growth factor receptor and activation of mitogen activated protein (MAP) kinases cascades. p21 blocks progression through the cycle and cell division by inactivating cyclin/cyclin-dependent kinase complexes. Preventing hyperphosphatemia and elevated Ca x P product in renal failure not only ameliorates the progression of SH and bone disease but also the morbidity and mortality resulting from vascular calcification.

A novel mechanism for skeletal resistance in uremia.

BACKGROUND: In treating secondary hyperparathyroidism, the target level of serum intact parathyroid hormone (I-PTH) should be three to five times normal to prevent adynamic bone disease. In circulation, there is a non-(1-84) PTH-truncated fragment, likely 7-84, which, in addition to PTH 1-84, is measured by most I-PTH immunoradiometric (IRMA) assays, giving erroneously high I-PTH values. We have developed a new IRMA assay in which the labeled antibody recognizes only the first six amino acids of the PTH molecule. Thus, this new IRMA assay (Whole PTH) measures only the biologically active 1-84 PTH molecule. METHODS: Using this new IRMA assay (Whole PTH) and the Nichols "intact" PTH assay, we compared the ability of each assay to recognize human PTH (hPTH) 1-84 and hPTH 7-84 and examined the percentage of non-1-84 PTH in circulation and in parathyroid glands. Possible antagonistic effects of the 7-84 PTH fragment on the biological activity of 1-84 PTH in rats were also tested. RESULTS: In 28 uremic patients, PTH values measured with the Nichols assay, representing a combined measurement of both hPTH 1-84 and hPTH 7-84, were 34% higher than with the Whole assay (hPTH 1-84 only); the median PTH was 523 versus 318 pg/mL (P < 0.001). Similar results were found in 14 renal transplant patients. In osteoblast-like cells, ROS 17.2, 1-84 PTH (10-8 mol/L) increased cAMP from 18.1 +/- 1.25 to 738 +/- 4.13 mmol/well. Conversely, the same concentration of 7-84 PTH had no effect. In parathyroidectomized rats fed a calcium-deficient diet, 7-84 PTH was not only biologically inactive, but had antagonistic effects on 1-84 PTH in bone. Plasma calcium was increased (0.65 mg/dL) two hours after 1-84 PTH treatment, while 7-84 PTH had no effect. When 1-84 PTH and 7-84 PTH were given simultaneously in a 1:1 molar ratio, the calcemic response to 1-84 PTH was decreased by 94%. In normal rats, the administration of 1-84 PTH increased renal fractional excretion of phosphate (11.9 to 27.7%, P < 0.001). However, when 1-84 PTH and 7-84 PTH were given simultaneously, the 7-84 PTH decreased the phosphaturic response by 50.2% (P < 0.005). Finally, in surgically excised parathyroid glands from six uremic patients, we found that 44.1% of the total intracellular PTH was the non-PTH (1-84), most likely PTH 7-84. CONCLUSION: In patients with chronic renal failure, the presence of high circulating levels of non-1-84 PTH fragments (most likely 7-84 PTH) detected by the "intact" assay and the antagonistic effects of 7-84 PTH on the biological activity of 1-84 PTH explain the need of higher levels of "intact" PTH to prevent adynamic bone disease.

Vitamin D.

The vitamin D endocrine systems plays a critical role in calcium and phosphate homeostasis. The active form of vitamin D, 1, 25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], binds with high affinity to a specific cellular receptor that acts as a ligand-activated transcription factor. The activated vitamin D receptor (VDR) dimerizes with another nuclear receptor, the retinoid X receptor (RXR), and the heterodimer binds to specific DNA motifs (vitamin D response elements, VDREs) in the promoter region of target genes. This heterodimer recruits nuclear coactivators and components of the transcriptional preinitiation complex to alter the rate of gene transcription. 1,25(OH)(2)D(3) also binds to a cell-surface receptor that mediates the activation of second messenger pathways, some of which may modulate the activity of the VDR. Recent studies with VDR-ablated mice confirm that the most critical role of 1, 25(OH)(2)D(3) is the activation of genes that control intestinal calcium transport. However, 1,25(OH)(2)D(3) can control the expression of many genes involved in a plethora of biological actions. Many of these nonclassic responses have suggested a number of therapeutic applications for 1,25(OH)(2)D(3) and its analogs.

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.

Pathogenesis of secondary hyperparathyroidism.

Secondary hyperparathyroidism is a universal complication in patients with chronic renal failure. Hyperplasia of the parathyroid glands is typically seen in these patients. In early renal failure, alteration in vitamin metabolism, decreased levels of calcitriol and moderate decreases in ionized calcium may allow greater synthesis and secretion of PTH. As the disease progresses, there is a decrease in the number of vitamin D receptors (VDR) and calcium receptors (CaR). The decreased number of VDR and CaR makes the parathyroid glands more resistant to calcitriol and calcium. Phosphorus induces hyperplasia of the parathyroid glands independent of calcium and calcitriol, and by a post-transcriptional mechanism increases PTH synthesis and secretion. Experimental work in uremic rats demonstrated that if the animals are fed a high-phosphorus diet, they not only developed secondary hyperparathyroidism but parathyroid cell hyperplasia. If the diet is then reduced in phosphorus, the levels of PTH return to normal. However, the parathyroid cell hyperplasia persists and no apoptosis is seen. Thus, the control of the three most important factors, calcium, calcitriol and phosphorus, is critical to prevent the development of secondary hyperparathyroidism and hyperplasia of the parathyroid glands.

New analogs of vitamin D3.

Calcitriol, the most active metabolite of vitamin D, controls parathyroid gland growth and suppresses the synthesis and secretion of parathyroid hormone (PTH). However, because of its potent effects on intestinal calcium absorption and bone mobilization, calcitriol treatment can induce hypercalcemia, often precluding its use at therapeutic doses. Hyperphosphatemia is also a persistent problem among patients undergoing chronic hemodialysis and can be aggravated by therapeutic doses of calcitriol. Several pharmaceutical companies were able to modify the side-chain of the 1,25(OH)2D3, allowing some of these new analogs to retain the action on the parathyroid glands while decreasing their hypercalcemic and hyperphosphatemic effects. The structure-activity relationship for ligand-mediated transcriptional regulation has been studied in detail. In some analogs the serum binding protein (DBP) plays a key role in determining the pharmacokinetics of the vitamin D compound. The affinity to DBP for 22-oxacalcitriol (OCT), an analog of calcitriol for the treatment of secondary hyperparathryoidism, is approximately 300-400 times lower than that of calcitriol and the analog is rapidly cleared from the circulation. The mechanisms for the selectivity of 19-nor-1,25(OH)2D2 (paricalcitol) (Zemplar) another analog of calcitriol, is clearly different from OCT. Although the mechanisms of action is not completely known, it does appear that paricalcitol down-regulates the VDR in the intestine. It is likely that the unique biological profiles of vitamin D analogs in vivo are due to multiple mechanisms. Understanding the molecular basis of the analog selectivity will not only provide an explanation for their unique actions but allow intelligent design of more effective analogs in the future.

Mechanism of vitamin D action and its regulation.

Most of the biological actions of 1,25(OH)2D3, the hormonal form of vitamin D, are mediated by the vitamin D receptor (VDR), a member of the steroid/thyroid receptor superfamily. The VDR functions as a ligand-induced transcription factor regulating the rate of expression of genes involved not only in the control of calcium homeostasis and bone remodeling, but also in immunomodulation, the control of hormone secretion, Inhibition of cell growth, and induction of cell differentiation. This section discusses the molecular mechanisms involved in the transcriptional control of gene expression by 1,25(OH)2D3.

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.

gamma-Interferon-induced resistance to 1,25-(OH)2 D3 in human monocytes and macrophages: a mechanism for the hypercalcemia of various granulomatoses.

The hypercalcemia of various granulomatoses is caused by endogenous 1,25-dihydroxyvitamin D [1,25-(OH)2D3] overproduction by disease-activated macrophages. The inability of 1,25(OH)2D3 to suppress its synthesis in macrophages contrasts with the tight control of its production in macrophage precursors, peripheral blood monocytes (PBM). We examined whether 1,25(OH)2D3 resistance develops as PBM differentiate to macrophages or with macrophage activation. Normal human pulmonary alveolar macrophages (PAM) are less sensitive to 1,25(OH)2D3 than PBM, despite similar vitamin D receptor content; however, both PBM and PAM respond to exogenous 1,25-(OH)2D3 by inhibiting 1,25(OH)2D3 synthesis and inducing 1,25(OH)2D3 degradation through enhancement of 24-hydroxylase mRNA levels and activity. The human monocytic cell line THP-1 mimics PAM in 1,25(OH)2D3 synthesis and sensitivity to exogenous 1,25(OH)2D3. We utilized THP-1 cells to examine the response to 1,25(OH)2D3 with macrophage activation. Activation of THP-1 cells with gamma-interferon (gamma-IFN) enhances 1,25(OH)2D3 synthesis 30-fold, blocks 1,25-(OH)2D3 suppression of its synthesis, and reduces by 42.2% 1,25-(OH)2D3 induction of its degradation. The antagonistic effects of gamma-IFN are not merely restricted to enzymatic activities. In THP-1 cells and in normal PBM, gamma-IFN inhibits 1,25-(OH)2D3 induction of 24-hydroxylase mRNA levels without reducing mRNA stability, suggesting gamma-IFN inhibition of 1,25(OH)2D3 transactivating function. These results explain 1,25(OH)2D3 overproduction in granulomatoses and demonstrate potent inhibition by gamma-IFN of 1,25(OH)2D3 action in immune cells.

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.

1,25-Dihydroxyvitamin D synthesis in rat liver microsomes.

Previous studies from our laboratory have shown 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] production by rat liver homogenates and a low affinity of the hepatic enzyme for 25-hydroxyvitamin D3. Because the liver microsomal vitamin D-25-hydroxylase is the main source of systemic 25(OH)D3, we examined the subcellular location and the kinetics of liver 1,25(OH)2D3 production. Unlike the renal 1 alpha-hydroxylase activity which was assayed simultaneously, 1,25(OH)2D3 synthesis was undetectable in rat liver mitochondria, whereas in microsomes, 1,25(OH)2D3 production followed typical Michaelis Menten kinetics with a Km for 25(OH)D3 of 13.4 microM and a Vmax of 109.8 pg/min per mg protein accounting for most of the 1,25(OH)2D3 synthesized by rat liver cytosol free homogenates. Thus, microsomes are the site for 1,25(OH)2D3 synthesis in the rat liver. This microsomal compartmentalization of the two major steps in the activation of vitamin D to 1,25(OH)2D3 suggests a role for the liver as an autocrine/paracrine organ for 1,25(OH)2D3.

Microtubules mediate cellular 25-hydroxyvitamin D3 trafficking and the genomic response to 1,25-dihydroxyvitamin D3 in normal human monocytes.

The genomic actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the intracellular vitamin D receptor (VDR). Although immunocytochemistry has shown that disruption of microtubular assembly prevents nuclear access of the sterol-VDR complex, the role of microtubules in the response to 1,25(OH)2D3 has not been studied in viable cells. Our studies examined this interaction in normal human monocytes. Monocytes convert 25(OH)D3 to 1,25(OH)2D3 and to 24-hydroxylated metabolites more polar than 1,25(OH)2D3. Microtubule disruption totally abolished the ability of exogenous 1,25(OH)2D3 to suppress its own synthesis and to induce 24-hydroxylase mRNA and activity, without affecting either total 1,25(OH)2D3 uptake or maximal 1,25(OH)2D3-VDR binding. Thus, intact microtubules are essential for 1,25(OH)2D3-dependent modulation of gene transcription. Interestingly, microtubule disruption also decreased monocyte 1,25(OH)2D3 synthesis, not by decreasing the Vmax of monocyte mitochondrial 1 alpha-hydroxylase but through an increase in the Km for 25(OH)2D3. We examined 25(OH)D3 transport. Microtubule disruption did not affect total cellular 25(OH)D3 uptake but reduced its intracellular trafficking to the mitochondria. Thus, microtubules participate in intracellular 25(OH)D3 transport, and their integrity determines normal 1,25(OH)2D3 synthesis.

Kinetics of monocyte 1 alpha-hydroxylase in renal failure.

In chronic uremia, the requirement of supraphysiological doses of serum 25-hydroxyvitamin D3 [25(OH)D3] for the normalization of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels has been attributed to impaired substrate availability to renal 1 alpha-hydroxylase. Because serum 1,25(OH)2D3 can also be corrected by 25(OH)D3 supplementation in bilaterally nephrectomized patients, we examined the role of substrate availability on 1,25(OH)2D3 production by peripheral blood monocytes (PBM). In hemodialysis patients (HP), 25(OH)D3 uptake was 50% lower than normal, and the maximal velocity (Vmax) and apparent Michaelis constant (Km) for 25(OH)D3 of 1 alpha-hydroxylase were 2.7- and 4-fold above normal, respectively. When serum 1,25(OH)2D3 of HP was corrected by intravenous 1,25(OH)2D3, 25(OH)D3 uptake, Km, and Vmax returned to normal values. The effect of 25(OH)D3 supplementation was also examined. In normal adults, 25(OH)D3 administration had no effect on serum 1,25(OH)2D3 levels nor on the Km or the Vmax of PBM 1 alpha-hydroxylase but caused a 11-fold increase in serum 24R,25-dihydroxyvitamin D3[24R, 25(OH)2D3]. In HP, 25(OH)D3 therapy raised serum 1,25(OH)2D3 and reduced the Km and Vmax of PBM 1 alpha-hydroxylase, which correlated negatively with serum 1,25(OH)2D3. However, serum 24R,25(OH)2D3 only increased slightly above basal. These results demonstrate that, in HP, 1) impaired uptake of 25(OH)D3 and low affinity for substrate determine the need for high 25(OH)D3 levels to normalize serum 1,25(OH)2D3, despite higher enzymatic activity; 2) 1,25(OH)2D3 deficiency plays a role in enhanced 1,25(OH)2D3 synthesis and impaired access of 25(OH)D3 to PBM 1 alpha hydroxylase; and 3) abnormal 25(OH)D3 delivery also affects 24-hydroxylation.

Modulation of renal osteodystrophy by extrarenal production of calcitriol.

We report a patient with severe chronic renal failure who developed spontaneous bone fractures. He was found to have hypercalcemia, normal calcitriol levels (probably due to extrarenal production by noncaseating granulomas), and functional hypoparathyroidism. The bone biopsy showed low bone turn-over and the presence of noncaseating granulomas. Treatment with corticosteroids decreased the calcium and calcitriol levels and the parathyroid hormone levels rose. No further fractures occurred. A repeat bone biopsy revealed the presence of osteitis fibrosa. Renal osteodystrophy may be modulated by extrarenal production of calcitriol. In this case, excessive suppression of parathyroid hormone by endogenous calcitriol presumably caused an adynamic bone lesion and spontaneous fractures.