Angiotensin II increases parathyroid hormone-related protein (PTHrP) and the type 1 PTH/PTHrP receptor in the kidney

Angiotensin II (AngII) participates in the pathogenesis of kidney damage. Parathyroid hormone (PTH)-related protein (PTHrP), a vasodilator and mitogenic agent, is upregulated during renal injury. The aim of this study was to investigate the potential relation between AngII and PTHrP system in the kidney. Different methods were used to find that both rat mesangial and mouse tubuloepithelial cells express PTHrP and the type 1 PTH/PTHrP receptor (PTH1R). In these cells, AngII increased PTHrP mRNA and protein production. In contrast, PTH1R mRNA was increased in mesangial cells and downregulated in tubular cells, but its protein levels were unmodified in both cells. AT(1) antagonist, but not AT(2), abolished AngII effects on PTHrP/PTH1R. The in vivo effect of AngII was further investigated by systemic infusion (a low dose of 50 ng/kg per min) into normal rats. In controls, PTHrP immunostaining was mainly detected in renal tubules. In AngII-infused rats, PTHrP staining increased in renal tubules and appeared in the glomerulus and the renal vessels. After AngII infusion, PTHR1 staining was markedly increased in all these renal structures at day 3 but remained elevated only in tubules at day 7. The AT(1) antagonist, but not the AT(2), significantly diminished AngII-induced PTHrP and PTHR1 overexpression in the renal tissue, associated with a decrease in tubular damage and fibrosis. The results indicate that AngII regulates renal PTHrP/PTH1R system via AT(1) receptors. These findings demonstrate that PTHrP upregulation occurs in association with the mechanisms of AngII-induced kidney injury.     

硝苯地平和罗钙全对尿毒症患者甲状旁腺素受体基因表达的影响

目的 观察钙通道阻滞剂硝苯地平和活性维生素Ｄ制剂罗钙全对尿毒症患者外周血单个核细胞（ＰＢＭＣ）上甲状旁腺素（ＰＴＨ）受体基因表达的影响。方法 ３０例非糖尿病常规血液透析患者随机分成硝苯地平组、罗钙全组和安慰剂组,治疗８周。应用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）方法检测用药前后ＰＢＭＣ上ＰＴＨ受体ｍＲＮＡ表达。结果 硝苯地平治疗能使尿毒症患者ＰＢＭＣ上ＰＴＨ受体ｍＲＮＡ的低表达显著上调（０．１４３     

PTHrP enhances the secretory response of PTH to a hypocalcemic stimulus in rat parathyroid glands

BACKGROUND: The secretion of parathyroid hormone (PTH) from the parathyroid glands might be regulated by autocrine/paracrine factors, and a feedback regulatory mechanism of PTH on the secretion of PTH has been suggested. Because of the existence of a common receptor between PTH and PTH-related peptide (PTHrP), the aim of the present study was to examine the possible effects of PTHrP 1-40 and 1-86 on PTH secretion in rats. METHODS: In vivo, the effect of PTHrP on Ca++-regulated PTH secretion was examined by the induction of hypocalcemia and hypercalcemia by an infusion of EGTA and Ca++, with and without PTHrP. The eventual effects of PTHrP on the peripheral metabolism of PTH were examined by infusion of human PTH (hPTH) with and without PTHrP. hPTH was measured by an intact hPTH assay not cross reacting with rat PTH or PTHrP. To examine whether near physiological levels of circulating PTH have an autoregulatory effect in vivo on PTH secretion from the parathyroid gland, an acute reduction of the circulating PTH was induced by an acute unilateral parathyroidectomy (UPTX). PTH secretion from the remaining parathyroid gland was followed in response to EGTA-induced hypocalcemia. In vitro investigations on the effect of PTHrP 1-40 on PTH secretion from whole rat parathyroid glands incubated in media containing a calcium concentration of 0.6 or 1.35 mmol/L were performed to confirm whether the effect of PTHrP was directly on the gland. The rat PTH assay was examined for cross reaction with PTHrP. RESULTS: In vivo, the same rate of decrease of plasma Ca++ was induced in the experimental groups. The maximal response of PTH to hypocalcemia (218 +/- 16 pg/mL, N = 6) was significantly enhanced by PTHrP 1-40 (525 +/- 79 pg/mL, N = 6) and by PTHrP 1-86 (465 +/- 29 pg/mL, N = 6, P < 0.001). No effect of PTHrP on PTH secretion was found during normocalcemia or hypercalcemia. UPTX resulted in a 50% reduction of PTH secretion, and no compensatory increase of PTH was observed. PTHrP had no effect on the metabolism of PTH. In vitro, low-Ca++-induced PTH secretion was significantly augmented by 300% (P < 0.01) when the medium contained PTHrP 1-40. PTHrP did not cross react with the PTH assay. CONCLUSIONS: PTHrP significantly enhanced the low-Ca++-stimulated PTH secretion in vivo and in vitro. An autocrine/paracrine role of PTHrP in the parathyroid glands is suggested. An autoregulatory effect of circulating PTH on the PTH secretion from parathyroid glands seems unlikely. The "maximal secretory capacity" of the parathyroid glands induced by hypocalcemia in vivo and in vitro is not the maximum, as PTH secretion can be increased even further, by several-fold.     

Chronic phosphorus supplementation decreases the expression of renal PTH/PTHrP receptor mRNA in rats

Dietary intake of high levels of phosphorus is known to increase serum levels of parathyroid hormone (PTH); however, how this increased serum PTH affects the action of PTH in major target tissues, particularly by kidney, remains unknown. In the present study, we therefore undertook to clarify this point in intact animals fed a high-P diet by examining various parameters of PTH action. Twelve weanling Wistar male rats were assigned randomly to two groups: a control group with dietary Ca:P = 1:1 and a high-P group (Ca:P = 1:3) fed the standard AIN-76 diet supplemented with P (0.5 and 1.5 g/100 g of diet). After 3 weeks of feeding, in the high-P diet group, we observed that serum Ca was lowered, without a difference in serum P, when compared to the control group. Excretion of urinary cAMP, an index of renal PTH action, was also decreased, with higher excretion of urinary P in those rats fed the high-P diet. In agreement with the decreased cAMP excretion, a clear reduction in PTH/PTH-related protein (PTHrP) receptor gene expression as estimated by Northern blotting was observed in the kidney, despite increased levels of serum PTH. Thus, the present study indicated that a high-P diet reduces PTH action in the kidney, though the serum PTH is increased.     

PTH/PTHrP receptor mRNA is down-regulated in epiphyseal cartilage growth plate of uraemic rats

PTH/PTHrP receptor mRNA is down-regulated in epiphyseal cartilagegrowth plate of uraemic rats. Growth retardation, hypocalcaemia,hyperphosphataemia, and skeletal resistance to the action ofPTH are well known features of advanced chronic renal failure(CRF). It has been suggested that the downregulation of renaland skeletal PTH receptors (PTH/PTHrP-R) could play an importantrole in the occurrence of these abnormalities. In the presentstudy, four uraemic (4 weeks after 5/6 nephrectomy) and fourcontrol (sham-operated) rats were analysed for PTH/PTHrP-R mRNAexpression at the proximal femoral and tibial growth platesby in situ hybridization. Uraemic rats had plasma biochemicalabnormalities of advanced CRF including high creatinine, phosphate,and PTH, and low calcium and calcitriol levels. The femoraland tibial bones of uraemic animals were shorter in length thanthose of control rats, and had reduced width and cellularityof the epiphyseal cartilage growth plate. Mean (±SD)tibia growth plate width was 152±30 µm in uraemicrats, compared with 170±35 µm in control rats.The difference was mostly due to a marked reduction of the zoneexpressing PTH/PTHrP-R (mature chondrocytes) which was 30±5µm in tibias from uraemic versus 44±10 µmin tibias from control rats. The hybridization signals of PTH/PTHrP-Rper individual cell were quantified on dark field images usinga computer-assisted image analysis system. The number of grainsin PTH/PTHrP-R positive cells was also decreased in uraemicrats, 103±13 compared with 123±14 arbitrary units(dark pixel density)/cell in control rats (P 0.005). In conclusion,these data indicate that rats with severe CRF and secondaryhyperparathyroidism have reduced epiphyseal cartil age PTH/PTHrP-RmRNA expression. This alteration may be relevant in the pathogenesisof growth retardation in uraemia.     

Decreased calcium-sensing receptor expression in hyperplastic parathyroid glands of uremic rats: role of dietary phosphate

BACKGROUND: The abnormal control of parathyroid hormone secretion in chronic renal failure is attributed, in part, to down-regulation of the calcium-sensing receptor (CaR) in hyperplastic parathyroid tissue. The cause of this down-regulation is unknown. Here we examined the roles of uremia and parathyroid hyperplasia on parathyroid gland (PTG) CaR expression in the rat model of renal failure. METHODS: Rats made uremic by 5/6 nephrectomy were maintained for one month on diets containing 0.2% P (low phosphate), 0.5% P (normal phosphate) or 1.2% P (high phosphate); intact rats (controls) were maintained on the normal-phosphate diet. RESULTS: CaR mRNA was reduced only in uremic rats fed the high-phosphate diet (55% less than in controls, P < 0.05). Immunohistochemical staining revealed decreased CaR protein expression in uremic high-phosphate rat PTG compared with controls (41% decrease as determined by computer-assisted quantitation, P < 0.01). PTG size was increased in uremic rats fed the high-phosphate diet compared with controls (2.77 +/- 0.95 vs. 0.77 +/- 0.16 microgram/g body wt, P < 0.0001). There was no increase in PTG size in uremic rats fed the low-phosphate and normal-phosphate diets (0.92 +/- 0.31 and 1.01 +/- 0.31 micrograms/g) compared with controls (0.77 +/- 0.16 microgram/g body wt). Immunohistochemical staining for proliferating cell nuclear antigen in hyperplastic PTG from uremic rats showed that CaR was decreased primarily in areas of active cell proliferation. CONCLUSION: These results suggest that CaR down-regulation cannot be attributed to uremia per se, but rather, is associated with parathyroid cell proliferation. Furthermore, dietary phosphate restriction prevents both the parathyroid hyperplasia and decreased CaR expression in renal failure.     

肾性甲状旁腺功能亢进患者甲状旁腺素钙敏感受体的表达

目的研究肾性甲状旁腺功能亢进(甲旁亢)患者甲状旁腺组织钙敏感受体(CaR)的表达,探讨CaR在肾性甲旁亢发病机制中的作用。方法用免疫组织化学的方法,比较正常对照组和继发性甲旁亢(SHPT)组甲状旁腺CaR蛋白质的表达。结果CaR在正常甲状旁腺组织的细胞膜和细胞浆均有表达,细胞阳性率为(75．20±2．31)％,而SHPT组为(27．88±4．90)％,明显减少(P＜0．01)。弥漫性增生和结节性增生之间差异也有显著性[分别为(40．00±3．34)％和(15．75±1．75)％,P＜0．01]。CaR表达阳性率与腺体重量呈负相关(r＝0．86,P＜0．01)。结论严重肾性甲旁亢患者甲状旁腺CaR表达明显下降,结节性增生比弥漫性增生下降更显著,是引起PTH过度分泌的重要原因。上调CaR的表达或激活CaR的功能将成为肾性甲旁亢新的治疗目标。     

PTH/PTHrP受体mRNA在血液透析患者成骨细胞中的表达

目的 了解PTH/PTHrP受体mRNA在维持性血液透析(HD)患者成骨细胞中的表达及钙离子拮抗剂(CCB)和骨化三醇对其影响。方法 21例随机分为HD试验组(8例)、骨化三醇治疗组(7例)及CCB治疗组(6例),并设置正常对照组(5例)。分别采取各例骨髓作成骨细胞培养。应用半定量RT-PCR方法观察各组成骨细胞的PTH/PTHrP受体mRNA表达。结果 与正常对照组比,HD患者PTH/PTHrP受体mRNA的表达下调(P<0.001),且与血中iPTH水平呈负相关(r=-0.673,P<0.001)。适量的骨化三醇可上调受体表达,而大剂量的骨化三醇可下调表达。CCB可上调HD患者的PTH/PTHrP受体表达。结论 HD患者成骨细胞PTH/PTHrP受体mRNA的表达下调。CCB上调PTH/PTHrP受体。适量的骨化三醇可以上调受体,而大剂量的骨化三醇抑制PTH和下调PTH/PTHrP受体。     

Down-regulation of human osteoblast PTH/PTHrP receptor mRNA in end-stage renal failure

BACKGROUND: Resistance to the action of parathyroid hormone (PTH) has been demonstrated in end-stage renal failure and is considered to be important in the pathogenesis of secondary hyperparathyroidism. The mechanism of resistance is unknown. However, altered regulation of cellular PTH/PTH-related protein (PTH/PTHrP) receptor (PTH1R) has been assumed to be important. METHODS: We have used in situ hybridization to examine PTH1R mRNA expression by osteoblasts in human bone and have compared the expression in high- and low-turnover renal bone disease, high-turnover nonrenal bone disease (healing fracture callus and Pagetic bone), and normal bone. Bone biopsies were formalin fixed, ethylenediaminetetraacetic acid decalcified, and paraffin wax embedded. A 1.8 kb PTH1R cDNA probe, labeled with 35S, was used, and the hybridization signal was revealed by autoradiography. The density of signal over osteoblasts was quantitated using a semiautomated Leica image analysis software package. RESULTS: The mean density of PTH1R mRNA signal over osteoblasts in renal high-turnover bone was only 36% of that found in nonrenal high-turnover bone (P < 0.05) and 51% of that found in normal bone (P < 0.05). Osteoblast PTH1R mRNA signal in adynamic bone from individuals with diabetes mellitus was 28% of normal bone (P < 0.05) and 54% of that found in renal high-turnover bone (P < 0.05). CONCLUSIONS: These results demonstrate a down-regulation of osteoblast PTH1R mRNA in end-stage renal failure in comparison to normal and high-turnover bone from otherwise healthy individuals, and provide an insight into the mechanisms of "skeletal resistance" to the actions of PTH.     

Development of craniofacial structures in transgenic mice with constitutively active PTH/PTHrP receptor

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) regulate calcium homeostasis, and PTHrP further regulates growth and development. A transgenic mouse carrying the constitutively active PTH/PTHrP receptor (HKrk-H223R) under the control of the mouse bone and odontoblast-specific alpha1(I) collagen promoter (Col1-caPPR) has been developed to demonstrate the complex actions of this mutant receptor in hard tissue formation. We have further characterized Col1-caPPR mice abnormalities in the craniofacial region as a function of development. Col1-caPPR mice exhibited a delay in embryonic bone formation, followed by expansion of a number of craniofacial bones including the maxilla and mandible, delay in tooth eruption and teratosis, and a disrupted temporomandibular joint (TMJ). These findings suggest that the Col1-caPPR mouse is a useful model for characterization of the downstream effects of the constitutively active receptor during development and growth, and as a model for development of treatments of human diseases with similar characteristics.     

肾脏病患者外周血淋巴细胞上甲状旁腺素受体的表达

目的　探讨人外周血淋巴细胞上有无甲状旁腺素（PTH）受体表达及其在肾脏疾病不同阶段的改变。方法应用RT-PCR方法观察各组患者淋巴细胞和肾脏组织中PTH受体mRNA表达。结果人淋巴细胞上可检出PTH受体基因表达；在慢性肾炎肾功能代偿期和尿毒症患者中均有不同程度下调，与肾功能损害密切相关，且与同组患者肾脏组织中PTH受体的改变相一致。结论肾功能减退时，患者淋巴细胞上PTH受体显著下调。     

肾脏病患者甲状旁腺素受体基因表达的研究

目的 探讨肾脏疾病不同阶段中甲状旁腺素(PTH)受体基因表达的变化及可能机制。方法 应用半定量RT/PCR方法观察各组患者肾穿刺或手术标本中PTH受体mRNA表达。结果 慢性肾炎肾功能正常、中度肾功能减退、尿毒症及急性肾衰患者均有不同程度PTH受体基因表达减少,且和肾脏损害密切相关。结论 在肾脏疾病进程中PTH受体下调明显早于肾功能、血PTH和钙磷的变化。     

选择性环氧化酶2抑制剂抑制尿毒症大鼠甲状旁腺异常增生

目的 评价环氧化酶2(COX2)抑制剂塞来昔布(celecoxib)对尿毒症大鼠甲状旁腺(PG)异常增生的影响.方法 通过5/6肾大部分切除结合高磷饮食(P 1.2％,Ca 1.2％)建立尿毒症甲状旁腺功能亢进症(甲旁亢)大鼠模型,存活大鼠按随机数字表法分成尿毒症甲旁亢非用药组(Nx-HP组,n=17)、塞来昔布预防组(Prey组,n=18,建模后1d起给塞来昔布100 mg· kg-1· d-1)和塞来昔布治疗组(Ther组,n=18,建模1个月后给塞来昔布100 mg·kg-1·d-1),并以假手术组作为对照(Sham组,n=14).12周后检测各组大鼠肾功能、血甲状旁腺激素(iPTH)水平、PG大小以及PG中增殖细胞核抗原(PCNA)、COX2表达.结果 Nx-HP组大鼠血清iPTH水平较Sham组显著升高[(100.73±4.35) ng/L比(34.77±0.83) ng/L,P＜0.01],塞来昔布干预后iPTH水平显著下降[Prev组(87.36±2.18) ng/L,Ther组(87.47±1.76) ng/L](均P＜ 0.05).计算显微镜下PG最大面积显示,Nx-HP大鼠PG最大面积显著增大,是Sham组的5.28倍[(2.436±0.372) mm2/kg比(0.461±0.089) mm2/kg,P＜0.01];而Prey组[(0.987±0.254)mm2/kg]和Ther组[(1.270±0.305) mm2/kg]PG分别缩小59.47％(P＜0.01)和47.87％ (P＜0.05),两组间差异无统计学意义.PCNA在Sham组PG中仅少量表达,Nx-HP组显著增多,塞来昔布干预后PCNA阳性细胞明显减少,Prey组和Ther组蛋白表达水平分别降低52.91％和34.68％(均P＜0.05).COX2在Sham组PG中几乎未见表达,但在尿毒症大鼠中明显增多,Nx-HP组、Prey组和Ther组分别为Sham组的2.47倍、2.34倍和3.04倍(均P＜0.05),而3组间差异无统计学意义.实时定量PCR检测PCNA和COX2基因水平显示同样变化趋势.结论 选择性COX2抑制剂塞来昔布可以显著抑制尿毒症大鼠甲旁亢和甲状旁腺增生.     

Decrease in the expression of the type 1 PTH/PTHrP receptor (PTH1R) on chondrocytes in animals with osteoarthritis

Background  

To evaluate the expression of the type 1 PTH/PTHrP receptor (PTH1R) on chondrocytes from hyaline cartilage over the course of osteoarthritis (OA).     

Mice expressing a constitutively active PTH/PTHrP receptor in osteoblasts show reduced callus size but normal callus morphology during fracture healing

Background The parathyroid hormone-/parathyroid hormone-related protein (PTH/PTHrP) receptor plays a crucial role in endochondral bone formation and possibly also in fracture healing. Patients with Jansen's metaphysial chondrodysplasia (JMC) have a gain-of-function mutation in the PTH/PTHrP receptor. Transgenic mice expressing JMC PTH/PTHrP receptor mutants in osteoblasts are characterized by increased trabecular bone formation and reduced osteoblastic activity at periosteal sites. We have analyzed the bone phenotype and studied the fracture healing process in this model.

Methods We performed bone density analysis of tibiae from 17-week-old transgenic mice and controls. Also, tibial fractures were produced in 14-week-old mice. Fracture healing was examined by radiographic and histological analysis.

Results Transgenic mice had a lower total bone mineral content (BMC), by a factor of one-third. The changes were bone compartment-specific with an increase in trabecular bone volume and a decrease in cortical thickness. The calluses in the transgenic mice were smaller, with a reduction in BMC and mean crosssectional area by a factor of one-half. Despite the smaller size, however, the morphology and progression through the healing process were similar in both transgenic and wild-type littermates.

Interpretation We conclude that the constitutively active PTH/PTHrP receptor has compartment-specific effects on bone formation when expressed in osteoblasts. During fracture healing, however, both the periosteal and the endochondral processes are activated, leading to fracture healing that is temporally and morphologically normal, although the callus tissue is less prominent.