1，25（OH）2D3与糖尿病肾病

我国2003年有2380万的糖尿病患者,2010年已超过了9200万,给国计民生带来沉重的负担.糖尿病肾病(DN)是糖尿病最常见的慢性并发症之一,也是导致终末期肾病(ESRD)的主要原因.目前,肾素血管紧张素系统(RAS)抑制剂、血管紧张素转换酶抑制剂(ACEI)和(或)血管紧张素受体阻滞剂(ARB)是治疗DN的一线药物,临床已证实这些药物能延缓肾小球硬化、小管问质纤维化和蛋白尿进展[1-2],然而,RAS抑制剂的1个主要问题是肾素逃逸,导致肾素代偿性增加[3-4].     

Lack of In vitro evidence for storage of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and 1,25(OH)2D3 binding protein in skeletal matrix

A few studies have reported on the measurement of 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃) in bone, using chloroform/methanol extraction and radioreceptor assay. As the significance of bone 1,25(OH)₂D₃ content was not defined in any of these reports, the objective of the current investigation was to determine whether 1,25(OH)₂D₃ may be stored in skeletal matrix. Bone powder samples from the iliac crest were extracted in ethylacetate/cyclohexane and 1,25(OH)₂D₃ isolated from the extract by means of Sephadex LH-20 and high pressure liquid chromatographic separation and subsequently measured by radioimmunoassay (RIA). Within the detection range of the RIA, no 1,25(OH)₂D₃ could be measured, suggesting that 1,25(OH)₂D₃ is not stored in skeletal matrix. Vitamin D bone concentrations previously measured may therefore have reflected plasma contamination. Consistent with this hypothesis, only traces of skeletal 1,25(OH)₂D₃ binding protein were measured when compared with serum values. Although 1,25(OH)₂D₃ may act as a potential local determinant of bone remodeling, there is no evidence supporting a delayed paracrine function by matrix-derived 1,25(OH)₂D₃.     

Oral 1,25(OH)2D3 pulse therapy

Conclusion In our experience, after a few months of therapy, every patient showed a marked improvement in both X-ray abnormalities derived from osteitis fibrosa and symptoms of renal osteodystrophy, especially bone pain, unless the serum phosphorus level was very high. The effectiveness of this therapy on the suppression of PTH secretion apparently depends on the initial PTH level, and also on the size of the gland itself. One of the major current difficulties in this therapy is the prevention of hypercalcemia when calcium carbonate is used. The calcium concentration of the dialysate must be reduced to 2.5 mEq/l not only for pulse therapy, but also for conventional therapy by vitamin D with calcium carbonate. Parathyroidectomy should be indicated only for the patient who does not respond to pulse therapy.     

Recruitment, augmentation and apoptosis of rat osteoclasts in 1,25-(OH)2D3 response to short-term treatment with 1,25-dihydroxyvitamin D3 in vivo

Background  

Although much is known about the regulation of osteoclast (OC) formation and activity, little is known about OC senescence. In particular, the fate of of OC seen after 1,25-(OH)₂D₃ administration in vivo is unclear. There is evidence that the normal fate of OC is to undergo apoptosis (programmed cell death). We have investigated the effect of short-term application of high dose 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) on OC apoptosis in an experimental rat model.     

1,25(OH)2D3 inhibits hormone secretion and proliferation but not functional dedifferentiation of cultured bovine parathyroid cells

Summary Increasing the extracellular Ca²⁺ concentration from 0.5 to 3.0 mM induced marked increments in cytoplasmic Ca²⁺ concentration (Ca²⁺ _i) and inhibition of parathyroid hormone (PTH) release of freshly isolated bovine parathyroid cells. 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃; 0.1–100 ng/ml) did not affect (Ca²⁺ _i) and was also without acute effect on the secretion. During 4 days of monolayer culture, the parathyroid cells underwent significant increases in both number and size, and presence of 10–100 ng/ml 1,25(OH)₂D₃ almost completely inhibited the cell proliferation, whereas the hypertrophy was unaffected. One day of culture with 0.1–100 ng/ml 1,25(OH)₂D₃ was without effect on PTH release but after 4 days there was a dose-related reduction of recretion. At this time point and irrespective of the culture condition, PTH release was no longer suppressed by high extracellular Ca²⁺. Furthermore, Ca²⁺ _i increased little upon increments in the extracellular Ca²⁺ concentration as compared with freshly isolated cells. It is concluded that after prolonged exposure to 1,25(OH)₂D₃, PTH release is inhibited and, at high concentrations, the parathyroid cells cease to proliferate. However, 1,25(OH)₂D₃ does not affect the development of functional dedifferentiation of parathyroid cells during monolayer culture.     

1,25(OH)2D3 receptor regulation and 1,25(OH)2D3 effects in primary cultures of growth cartilage cells of the rat

Summary Vitamin D deficiency leads to disturbed calcification of growth cartilage and enlargement of growth plate, illustrating that chondrocytes are a target for vitamin D. This observation prompted an investigation of 1,25(OH)₂D₃ receptor expression and action of vitamin D metabolites on chondrocyte proliferation. In primary cultures of tibial growth cartilage of male SD rats (80 g), specific binding of [³H]-1,25(OH)₂D₃ is noted in both the logarithmic growth phase and at confluence (N_max 12780 molecules/cell versus 4368 molecules/cell). Scatchard analysis revealed the presence of a single class of noninteracting binding sites. K_D was 10⁻¹¹ M irrespective of growth phase. The binding macromolecule had a sedimentation coefficient of 3.5 S. Interaction with DNA was demonstrated by DNA cellulose affinity chromatography. In immunohistology, growth cartilage cells (rabbit tibia) expressed nuclear 1,25(OH)₂D₃ receptors most prominently in the proliferative and hypertrophic zone. This corresponds to binding data which showed highest N_max in the proliferating cartilage. 1,25(OH)₂D₃ in the presence of delipidated fetal calf serum (FCS) had a biphasic effect on cell proliferation and density, i.e., stimulation at 10⁻¹² M and dose-dependent inhibition at 10⁻¹⁰ M and below. Inhibition was specific and not seen with 24,25(OH)₂D₃ or dexamethasone. Growth phase-dependent 1,25(OH)₂D₃ receptor expression and effects of 1,25(OH)₂D₃ on chondrocyte proliferation point to a role of vitamin D in the homeostasis of growth cartilage.     

1,25(OH)2D levels in dihydrotachysterol-treated patients: influence on 1,25(OH)2D assays

Many clinicians continue to prefer dihydrotachysterol (DHT) as the initial vitamin D agent of choice in hypoparathyroidism and renal osteodystrophy because of its long history of efficacy and safety. Assessment of the factors influencing the clinical response to DHT treatment should include measurement of vitamin D metabolite profiles, but investigators have heretofore been unable to measure 1,25(OH)2D because levels have been found to be falsely elevated when employing the chick intestinal cytosol receptor assay. After converting from the chick cytosol receptor assay to the calf thymus receptor assay for measuring 1,25(OH)2D, we did not note falsely elevated levels of 1,25(OH)2D in DHT-treated patients. The design of this study, therefore, was aimed at determining whether or not the calf thymus receptor measured authentic 1,25(OH)2D in such patients. We controlled for the possibility that freezing and thawing or prolonged storage might have either lowered 1,25(OH)2D levels or degraded a metabolite(s) of DHT that would have otherwise been recognized as "1,25(OH)2D" by the calf receptor. Similarly, technical differences between the two assays, source of thymus, and potential interference by other cytosolic proteins were eliminated as causes for the difference between the 1,25(OH)2D levels in the two assays. Our experiments do not provide an explanation for why the thymus receptor does not "see" the interfering metabolite(s) of DHT. This could reflect either a tissue difference or perhaps a species difference. Our results do provide the first opportunity to expand the investigation of the metabolic effects of DHT therapy to include changes in intrinsic 1,25(OH)2D metabolism.     

Metabolic effects of thiazide and 1,25-(OH)2 vitamin D in postmenopausal osteoporosis

It was previously shown that the stimulation of intestinal calcium absorption by exogenous 25-hydroxyvitamin D (25-OHD) in postmenopausal women with osteoporosis was attenuated when thiazide was added, probably due to the suppression of endogenous synthesis of 1,25-(OH)₂ vitamin D (1,25-(OH)₂D). To test whether the above attenuation could be averted if exogenous 1,25-(OH)₂D replaced 25-OHD, 10 women with postmenopausal osteoporosis participated in a three-phase study comprising control (pretreatment), treatment with 1,25-(OH)₂D 0.5 µg/day for 4 weeks, and combined 1,25-(OH)₂D and trichlormethiazide (TZ) 2 mg/day for 4 weeks. The 1,25-(OH)₂D treatment significantly increased serum 1,25-(OH)₂D from 60±7.2 (SD) to 154±48 pmol/1, fractional intestinal calcium absorption () from 0.386 ±0.055 to 0.613±0.081, and urinary calcium from 3.7±0.8 to 6.6±1.9 mmol/day. Addition of TZ significantly reduced urinary calcium from 6.6±1.9 to 4.8±1.3 mmol/day, without changing (0.613±0.081 to 0.584±0.070), serum calcium or 1,25-(OH)₂D (154±48 to 154±38 pmol/1). Thus, estimated calcium balance (absorbed minus urinary calcium, increased marginally to +5.6 mmol/day on 1,25-(OH)₂D alone (p=0.028) and significantly to +6.8 mmol/day on 1,25-(OH)₂D+TZ, from the control value of +4.0 mmol/day. Seven patients who were treated long-term with combined 1,25-(OH)₂D and TZ for 11–29 months maintained their a (0.593±0.099) and a marginally more positive estimated calcium balance (+6.4 mmol/day,p=0.025 from the control phase). Moreover, there was a stability of bone density of radial shaft, femoral neck, and lumbar spine. In conclusion, when exogenous 1,25-(OH)₂D is provided, TZ does not lower serum 1,25-(OH)₂D and in patients with postmenopausal osteoporosis. Thus, the hypocalciuric action of TZ may lead to improved calcium balance and may potentially attenuate further bone loss.     

Acidosis inhibits 1,25-(OH)2D3 but not cAMP production in response to parathyroid hormone in the rat

Parathyroid hormone (PTH) is a major activator of renal proximal tubule 25-hydroxyvitamin D3-1-hydroxylase (1-OHase). Chronic metabolic acidosis (CMA) inhibits 1-OHase and reduces circulating 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] levels in rats fed a low-Ca diet (LCD, 0.002% Ca). To examine the cellular mechanism whereby CMA inhibits 1-OHase, PTH-dependent renal 1-OHase activity and cAMP were measured in proximal tubules isolated from rats fed LCD for 14 days and made acidotic by the addition of 1.5% ammonium chloride to the drinking water. Serum 1,25-(OH)2D3 and proximal tubule 1-OHase activity and cAMP content were lower in acidotic rats. hPTH-(1-34) (10(-7) M) in vitro increased cAMP content to equivalent concentrations in tubules from rats with CMA and from nonacidotic controls; however, PTH increased 1-OHase activity only in tubules from nonacidotic animals. Although forskolin increased tubule cAMP content to equivalent levels in tubules from acidotic and nonacidotic rats, 1-OHase activity declined in tubules from nonacidotic rats and remained suppressed in acidotic tubules. The results suggest that chronic metabolic acidosis inhibits the PTH activation of 1-OHase through alteration of one or more steps in a cAMP-independent messenger system. PTH and forskolin can increase cAMP production by acidotic and nonacidotic proximal tubules; however, 1-OHase activity is not restored to normal in acidotic tubules and nonacidotic tubule 1-OHase may be inhibited.     

1,25(OH)2D and 24,25(OH)2D production in the developing kidney

To clarify the state of vitamin D production by the developing kidney, firstly, we measured serum levels of 1,25(OH)₂D and 24,25(OH)₂D in humans of different ages (pregnant and nonpregnant women, adult males, children and newborn infants) and secondly, we measured 1- and 24-hydroxylase activity in the kidney mitochrondria of rats at different ages. The mean serum levels of 1,25(OH)₂D in pregnant women, cord blood and newborns were significantly higher than those in children and non-pregnant women and adult males. In newborns, the level increased with gestational age. Synthesis of 1,25(OH)₂D was, at least in part, under the control of the fetus and newborn, rather then being solely a reflection of the conditions prevailing in the mother. The 1-hydroxylase activity in mitochondria was highest in the 1- to 2-month-old rats, and it decreased gradually thereafter. The change in 1-hydroxylase activity with age was due to a change in the V_max of the system.     

Association among plasma 1,25(OH)2D,ratio of 1,25(OH)2D to 25(OH)D,and prostate cancer aggressiveness

Background

African-American (AA) men tend to present with more aggressive prostate cancer (Gleason score >7) than European-American (EA) men. Vitamin D and its metabolites are implicated in prostate cancer biology with vitamin D deficiency, indicated by its metabolite levels in serum or plasma, usually observed in AA men.

Objective

To determine if 1, 25-dihydroxy vitamin D3 [1,25(OH)₂D] plasma levels in AA and EA prostate cancer patients alter the risk of having aggressive prostate cancer.

Design

Research subjects from the North Carolina-Louisiana Prostate Cancer Project (AA n = 435 and EA n = 532) were included. Plasma metabolites 1,25(OH)₂D and 25-hydroxyvitamin D3 [25(OH)D] were measured using liquid chromatography with tandem mass spectrophotometry. Research subjects were classified into low (Gleason sum < 7, stage T1-T2, and Prostate-specific antigen (PSA) < 9 ng/mL) or high (Gleason sum > 8 or Gleason sum = 7 with 4 + 3, or PSA > 20 ng/mL, or Gleason sum = 7 and stage T3-T4) aggressive disease.

Results

Research subjects in the second and third tertiles of plasma levels of 1, 25(OH)₂D had lower odds of high aggressive prostate cancer (AA [OR_T2vsT1: 0.66, 95%CI: 0.39-1.12; OR_T3vsT1: 0.83, 95%CI: 0.49-1.41] and EA [OR_T2vsT1: 0.68, 95%CI: 0.41-1.11; OR_T3vsT1: 0.67, 95%CI: 0.40-1.11]) compared with the first tertile, though confidence intervals included the null. Greater 1,25(OH)₂D/25(OH)D molar ratios were associated with lower odds of high aggressive prostate cancer more evidently in AA (OR_Q4vsQ1: 0.45, CI: 0.24-0.82) than in EA (OR_Q4vsQ1: 0.64, CI: 0.35-1.17) research subjects.

Conclusions

The 1,25(OH)₂D/25(OH)D molar ratio was associated with decreased risk of high aggressive prostate cancer in AA men, and possibly in EA men. Further studies analyzing vitamin D polymorphisms, vitamin D binding protein levels, and prostatic levels of these metabolites may be useful. These studies may provide a better understanding of the vitamin D pathway and its biological role underlying health disparities in prostate cancer.

     

Effect of 1,25(OH)2D3 and 24,25(OH)2D3 on calcium ion fluxes in costochondral chondrocyte cultures

Summary Vitamin D₃ metabolites have been shown to affect proliferation, differentiation, and maturation of cartilage cells. Previous studies have shown that growth zone chondrocytes respond primarily to 1,25(OH)₂D₃ whereas resting zone chondrocytes respond primarily to 24,25(OH)₂D₃. To examine the role of calcium in the mechanism of hormone action, this study examined the effects of the Ca ionophore A23187, 1,25(OH)₂D₃, and 24,25(OH)₂D₃ on Ca influx and efflux in growth zone chondrocytes and resting zone chondrocytes derived from the costochondral junction of 125 g rats. Influex was measured as incorporation of⁴⁵Ca. Efflux was measured as release of⁴⁵Ca from prelabeled cultures into fresh media. The pattern of⁴⁵Ca influx in unstimulated (control) cells over the incubation period was different in the two chondrocyte populations, whereas the pattern of efflux was comparable. A23187 induced a rapid influx of⁴⁵Ca in both types of chondrocytes which peaked by 3 minutes and was over by 6 minutes. Influx was greatest in the growth zone chondrocytes. Addition of 10⁻⁸–10⁻⁹ M 1,25(OH)₂D₃ to growth zone chondrocyte cultures results in a dose-dependent increase in⁴⁵Ca influx after 15 minutes. Efflux was stimulated by these concentrations of hormone throughout the incubation period. Addition of 10⁻⁶–10⁻⁷ M 24,25(OH)₂D₃ to resting zone chondrocytes resulted in an inhibition in ion efflux between 1 and 6 minutes, with no effect on influx during this period. Efflux returned to control values between 6 and 15 minutes.⁴⁵Ca influx was inhibited by these concentrations of hormone from 15 to 30 minutes. These studies demonstrate that changes in Ca influx and efflux are metabolite specific and may be a mechanism by which vitamin D metabolites directly regulated chondrocytes in culture.     

Oral high dose 1,25(OH)2D3 pulse therapy

To 13 uremic patients with secondary hyperparathyroidism, 4 micrograms of 1,25(OH)2D3 were given orally twice a week for 4 weeks. Intact PTH values fell from 488.3 +/- 84.2 to 235.2 +/- 59.6 pg/ml (Mean +/- SE, p less than 0.01), while serum total and ionized calcium elevated from 10.3 +/- 0.2 to 11.8 +/- 0.6 mg/dl (p less than 0.01), from 1.43 +/- 0.03 to 1.64 +/- 0.06 mmol/l (p less than 0.05), respectively, in 9 patients whose initial intact PTH level had been below 1000 pg/ml. The other 4 patients, of whom intact PTH level had been above 1000 pg/ml, did not show significant change in intact PTH values, though serum ionized calcium elevated slightly after this treatment. The correlation curve, determined by ionized calcium and intact PTH values in each period, was found to shift in only 2 out of 5. During the 4 weeks of high dose oral 1,25(OH)2D3 therapy, mean blood pressure elevated from 92.4 +/- 3.3 to 103.5 +/- 3.5 mmHg (p less than 0.01) in general, and 7 patients out of 13 complained of mental irritability. These data suggest that oral administration of high dose 1,25(OH)2D3 suppresses PTH secretion of uremic patients directly, however, reliability of this effect is still controversial. Indication of this therapy and adverse effects caused by rapid increase in serum calcium should be studied in more detail.     

Pharmacokinetics of 1,25(OH)(2)D(3) and 1alpha(OH)D(3) in normal and uraemic men.

BACKGROUND: The therapeutic equivalence of 1,25(OH)(2)D(3) and 1alpha(OH)D(3) on the suppression of PTH synthesis and secretion has not clearly been established. The aim of the present study was to evaluate the pharmacokinetics of 1,25(OH)(2)D(3) and 1alpha(OH)D(3) after oral and i.v. administration in healthy volunteers and uraemic patients. METHODS: Six healthy volunteers and 12 uraemic patients were included in the study. With an interval of 2 weeks, 4 microg of 1,25(OH)(2)D(3) i.v., 4 microg of 1,25(OH)(2)D(3) orally, 4 microg of 1alpha(OH)D(3) i.v. and 4 microg of 1alpha(OH)D(3) orally were administered. Blood samples for analysis of plasma-Ca(2+), plasma-1,25(OH)(2)D(3), and plasma-PTH were drawn at time 0, 0.25, 0.5, 1, 2, 4, 6, 9, 12, 24, 48, and 72 h. The healthy volunteers were studied in all four protocols and the uraemic patients in either the 1alpha(OH)D(3) (n=6) or the 1,25(OH)(2)D(3) (n=6) protocol. RESULTS: After oral administration of 1,25(OH)(2)D(3) the bioavailability of 1,25(OH)(2)D(3) was 70.6+/-5.8/72.2+/-4.8% in healthy volunteers/uraemic patients (n.s.). After i.v. administration the volume of distribution of 1,25(OH)(2)D(3) was similar, 0.49+/-0.14 vs 0.27+/-0.06 l/kg in healthy volunteers vs uraemic patients (n.s.), while the metabolic clearance rate of 1,25(OH)(2)D(3) was 57% lower in the uraemic patients, 23.5+/-4.34 vs 10.1+/-1.35 ml/min in healthy volunteers vs uraemic patients, respectively (P<0.03). The bioavailability of 1,25(OH)(2)D(3) after i.v. administration of 1alpha(OH)D(3) was 42.4+/-11.0/42.0+/-2.0% in healthy volunteers/uraemic patients (n.s.); and after oral administration of 1alpha(OH)D(3) 42.0+/-2.0/29.8+/-3.1% in healthy volunteers/uraemic patients (n.s.). A small, but significant increase in plasma-Ca(2+) was seen after administration of 1,25(OH)(2)D(3) to the uraemic patients, while no increase was seen after administration of 1alpha(OH)D(3). PTH levels were significantly suppressed in the healthy volunteers 24 h after administration of 4 microg of 1,25(OH)(2)D(3) i.v., 4 microg of 1,25(OH)(2)D(3) orally, and 4 microg of 1alpha(OH)D(3) orally by 35+/-7, 30+/-8, and 35+/-4%, respectively (all P<0.03). In the uraemic patients, PTH levels were significantly suppressed after administration of 4 microg of 1,25(OH)(2)D(3) i.v., 4 microg of 1,25(OH)(2)D(3) orally, and 4 microg of 1alpha(OH)D(3) i.v. by 30+/-10, 45+/-7, and 40+/-7%, respectively (all P<0.04). The effect was transitory in the healthy volunteers and lasted for at least 72 h in the uraemic patients. CONCLUSION: The present study found a 57% lower metabolic clearance rate of 1,25(OH)(2)D(3) in uraemic patients, as compared with that of healthy volunteers (P<0.03). The bioavailability of 1,25(OH)(2)D(3) following administration of 1alpha(OH)D(3) i.v. and orally in both healthy volunteers and uraemic patients was markedly lower than after administration of oral 1,25(OH)(2)D(3) (P<0.03). In spite of lower plasma-1,25(OH)(2)D(3) levels after administration of 1alpha(OH)D(3), no significant difference was observed on the suppressive effect of 4 microg i.v. of either 1,25(OH)(2)D(3) or 1alpha(OH)D(3) on the plasma-PTH levels in the uraemic patients. This might suggest the existence of an effect of 1alpha(OH)D(3) on the parathyroid glands which is independent of the plasma-1,25(OH)(2)D(3) levels, that are achieved after oral or i.v. administration of 1alpha(OH)D(3).     

Diminished parathyroid 1,25(OH)2D3 receptors in experimental uremia

In Sprague Dawley rats, six days after subtotal nephrectomy, serum 1,25(OH)2D3 concentration was diminished (59.8 +/- 17.5 pg/ml vs. 121 +/- 48; P less than 0.01). Despite low circulating 1,25(OH)2D3 levels, maximal specific binding capacity for 1,25(OH)2D3 in parathyroid glands was diminished (Nmax 87.5 fmol/mg protein and 3.52 fmol/mg DNA vs. 143 fmol/mg protein and 4.75 fmol/mg DNA, respectively). There was no change of KD, apparent molecular size (sucrose density gradient) and DNA binding affinity (DNA cellulose chromatography) pointing to intactness of the receptor. Since 1,25(OH)2D3 is a potent negative feedback signal for parathyroids, the data are potentially relevant for the genesis of secondary renal hyperparathyroidism.     

1,25(OH)2D deficiency induces temporomandibular joint osteoarthritis via secretion of senescence-associated inflammatory cytokines

1,25-Dihydroxyvitamin D [1,25(OH)₂D] insufficiency appears to be associated with several age-related diseases. Insufficient levels of serum 25-hydroxyvitamin D has been shown to lead to the progression of osteoarthritis (OA) while underlying biological mechanisms remain largely unknown. In this study, we sought to determine whether 1,25(OH)₂D deficiency has a direct effect on the process of murine temporomandibular joint (TMJ) OA in 25-hydroxyvitamin D 1α-hydroxylase knockout [1α(OH)ase^−/−] mice that had been fed a rescue diet (high calcium, phosphate, and lactose) from weaning until 6 or 18 months of age. Our results showed that the bone mineral density and subchondral bone volume were reduced in mandibular condyles, articular surfaces were collapsed, the thickness of articular cartilage and cartilage matrix protein abundance were progressively decreased and eventually led to an erosion of articular cartilage of mandibular condyles. We also found that DNA damage, cellular senescence and the production of senescence-associated inflammatory cytokines were increased significantly in 1α(OH)ase^−/− mice. This study demonstrates that 1,25(OH)₂D deficiency causes an erosive TMJ OA phenotype by inducing DNA damage, cellular senescence and the production of senescence-associated inflammatory cytokines. Our results indicate that 1,25(OH)₂D plays an important role in preventing the development and progression of OA.