CNP拮抗FGF-23/MAPK信号通路对肾性骨病的治疗效应及机制研究

Renal osteodystrophy (ROD) is one of the main causes of growth retardation, vascular calcification, spontaneous fracture and extremity disability in pediatric patients with nephropathies, usually characterized by insidious onset, slow progression and poor prognosis. Fibroblast growth factor-23 (FGF-23) has a significant inhibitory action on osteogenetic process, mediated by mitogen-activated protein kinase (MAPK). Increased serum FGF-23 level is present as early as the second stage of chronic kidney disease and has a good coincidence with the ROD onset.In our previous study, we found that C-type natriuretic peptide (CNP) served as a positive regulator of bone formation and its expression declined with the progression of chronic kidney disease. In this context, we hypothesize that FGF-23 may be an early marker of ROD, and antagonistic effect of CNP on the FGF-23/MAPK pathway may be a promising treatment option for ROD. The present study will first be conducted in adriamycin-induced nephrotic rats to research on the associations of serum FGF-23 with bone metabolic markers and bone pathology. Subsequently, the expression and phosphorylation of both the upstream and downstream signaling molecules of the FGF-23/MAPK pathway were determined in osteoblasts and adriamycin-induced nephrotic rats after treatment with exogenous CNP, by real-time PCR, immunofluorescence and western blot. The present study will provide the experimental evidence for improving the early diagnosis, evaluation and management of ROD.

肾性骨病（ROD）起病隐匿、进展缓慢、预后严重，是导致肾病患儿生长发育落后、血管钙化、自发性骨折和肢体残疾的主要原因。成纤维细胞生长因子-23（FGF-23）经丝裂原活化蛋白激酶 (MAPK) 介导对成骨过程具有显著的抑制作用，其血清浓度在慢性肾病2期开始升高，与ROD起病高度吻合。既往研究中，我们发现C型利钠肽（CNP）具有显著的促成骨效应，其表达随肾病进展逐渐衰减。由此推测：FGF-23可能是ROD起病早期的生物学标记；CNP拮抗FGF-23/MAPK通路有望为ROD治疗另辟蹊径。本课题首先研究阿霉素肾病大鼠血清FGF-23与骨代谢指标及骨组织病理相关性；在此基础上，运用实时定量PCR、免疫荧光、Western blot等方法探讨外源性CNP对体外培养成骨细胞及阿霉素肾病大鼠FGF-23/MAPK通路上、下游信号分子表达及磷酸化水平的影响，为ROD早期诊断、病情评估与治疗提供实验依据。

目的：肾性骨病(ROD)是导致肾病患儿生长发育落后、血管钙化、自发性骨折和肢体残疾的主要原因。成纤维细胞生长因子-23(FGF-23)经丝裂原活化蛋白激酶(MAPK)介导对成骨过程具有显著的抑制作用，其血清浓度在慢性肾病2期开始升高，与ROD起病高度吻合。既往研究中，我们发现C型利钠肽(CNP)具有显著的促成骨效应，其表达随肾病进展逐渐衰减。由此推测：FGF-23可能是ROD起病早期的生物学标记；CNP拮抗FGF-23/MAPK通路有望为ROD治疗另辟蹊径。方法：采用单侧肾切除联合尾静脉注射阿霉素构建大鼠ROD模型，治疗组给予外源性CNP静脉滴注，分别于造模后24h、72h、1w、2w、3w、1m、2m和3m处死，观察ROD大鼠血清FGF-23浓度与骨代谢指标及骨组织学改变，探讨CNP对FGF-23的抑制效应、对ROD的治疗作用、对ROD大鼠骨组织FGF-23/MAPK通路信号分子的作用机制。在大鼠成骨细胞培养体系中加入CNP(10和100pmol/l)，分别于24h、48h和72h后观察CNP对细胞增殖及骨胶原X(Col-X)表达和骨代谢指标的作用、对FGF-23/MAPK信号通路的作用。结果：(1)ROD大鼠血清FGF-23与血清钙，磷，25羟维生素D[25-(OH)D]，骨钙蛋白(OC)，总碱性磷酸酶(tAP)，骨特异性碱性磷酸酶(bAP)，I型前胶原羧基末端(PICP)，I型胶原交联羧基末端肽(ICTP)，耐酒石酸酸性磷酸酶(TRAP)，尿吡啶啉(PYD)，脱氧吡啶啉(DPD)和骨组织Col-X蛋白表达呈显著负相关；与骨组织FGF-23蛋白表达和血清甲状旁腺激素(PTH)呈显著正相关。(2)CNP可显著改善ROD大鼠的肾功能不全、钙磷代谢紊乱、低维生素D血症、继发性甲状旁腺功能亢进及低转化型骨代谢不良。CNP可诱导ROD大鼠骨组织中Klotho、FGFR1及旁路信号因子(STAT1/p-STAT1)的高表达，下调骨组织中FGF-23、RAF-1及其下游信号分子(ERK/p-ERK和P38/p-P38)和Col-X的表达；(3)CNP可显著促进成骨细胞增殖和Col-X表达。CNP可同时上调成骨指标(OC、PICP、tAP和bAP)和破骨指标(TRAP和ICTP)表达，显著下调成骨细胞FGF-23表达，而FGFR1和Klotho表达未见明显差异。