| ObjectiveOsteoporosis is a common complication in patients with chronic kidney disease(CKD),which is an important risk factor for fracture,disability and mortality.It is generally believed that 75% to 100% of patients will suffer from osteoporosis to some extent when GFR is less than 60 ml/min.Epidemiological studies found that the fracture risk of CKD patients was 4.4-14 times higher than that of non-CKD patients.Therefore,it was of great significance to explore the pathogenesis of osteoporosis in CKD.There were a lot of evidence that osteoporosis in CKD was related to increased bone marrow adipose.Recent studies have found that endothelial-to-mesenchymal transition(End MT)can occur in endothelial cells(ECs)under certain conditions,which can further differentiate into adipocytes.Therefore,endothelial-to-adipocyte transition might be a new mechanism of osteoporosis in CKD.Ciacalcet(CINA)is a commonly used drug for the treatment of CKD with secondary hyperparathyroidism(SHPT),which can significantly reduce the level of serum parathyroid hormone(PTH).Recent studies have found that CINA could also improve osteoporosis in patients with CKD,but the underlying mechanism was still unclear.Therefore,the purpose of this study was to explore the mechanism of CINA in improving CKD osteoporosis and its effect on endothelial-to-adipocyte transition,so as to provide theoretical basis for clinical prevention and treatment of CKD osteoporosis.MethodsThis study is divided into two parts.Part ? was in vivo study,part ? was in vitro study.Part ?: Study the effect of CINA on osteoporosis in CKD rats.Rats were divided into three groups: control group,CKD group and CKD + CINA group.The rats models in CKD group were constructed by feeding 0.75% adenine and high phosphorus diet(1.5%).At the age of 42-week-old,rats were sacrificed.Bone mineral density detection and micro-CT analysis were performed before sacrifice;Blood and bone tissues were collected after sacrifice for further blood biochemistry and bone metabolism analysis(bone mechanics,oil red O and HE staining).Endothelial cell markers CD31,mesenchymal cell markers(?-SMA and FSP1),mesenchymal stem cell markers(CD10 and CD44),adipocyte markers(PPAR-? and C/EBP-?)were detected by western blot.Bone marrow co-expression of CD31 and FSP1,CD31 and CD44 were also determined by immunofluorescence.Part ?: Exploring the effects of PTH on endothelial-to-adipocyte transition and the role of Wnt/?-catenin pathway.Human aortic endothelial cells were cultured in vitro,and the changes of End MT-related markers(CD31,FSP1 and ?-SMA)and nuclear translocation of ?-catenin expression were observed by western blot and immunofluorescence at different concentrations and time.The adipocyte markers(PPAR-? and C/EBP-?)were detected by western blot and the lipid droplets were determined by oil red O staining after a week of induction in adipocyte culture medium;Blocking the canonical Wnt/?-catenin signaling pathway with ?-catenin si RNA and DKK1 to detect the effects of PTH on End MT-related markers and further adipogenesis.ResultsIn Part ?,we demonstrated that the levels of serum creatinine,phosphorus and PTH in CKD group were significantly increased,blood calcium were markedly decreased compared with the control group.In addition,bone mineral density of lumbar vertebraes and femurs in CKD group decreased compared with the control group.The results of micro-CT showed that there were severe femoral cortical and cancellous bone loss in CKD group compared with the control group.The mechanical properties of CKD rats were significantly weakened compared with the control group,which characteried as decreased ultimate force,work to failure,stiffness and elastic modulus.The bone marrow fat were increased in the CKD group than the control group by HE stain and oil red O stain.CINA treatment could significantly reduce serum PTH,increase bone mineral density,alleviate trabecular and cortical bone loss,enhance bone mechanics,and reduce bone marrow adipose tissue infiltration in CKD rats.In addition,the levels of End MT-related markers in bone marrow of rats in CKD group were significantly higher than those in control group,which were significantly decreased after CINA treatment.The levels of serum PTH were correlated with the protein expression of End MT-related markers in bone marrow in each group.In Part ?,it was shown that PTH decreased the protein expression of CD31,and increased the expression of FSP1 and ?-SMA in a concentration and time dependent manner.Confocal microscopy revealed an increasing overlap of CD31 with FSP1 in some endothelial cells after PTH treatment.After one week of adipocyte differentiation,the expression of PPAR-? and C/EBP-? in PTH group was significantly higher than that in control group,and oil red O staining was positive.In addition,PTH treatment increased the expression of nuclear translocation of ?-catenin in a concentration-dependent manner.DKK1 and ?-catenin si RNA could significantly down-regulate the expression of PTH-induced End MT and subsequent PPAR-? and C/EBP-? expression.Conclusion1.CINA treatment could reduce serum PTH level,alleviate bone marrow fat,and improve osteoporosis in CKD rats;2.CINA treatment could reduce bone marrow End MT;3.Elevated PTH could promote End MT and further differentiate into adipocytes by activating Wnt/?-catenin signaling pathway.The PTH-induced endothelial-to-adipocyte transition might provide a new mechanism for bone loss in CKD;4.CINA might attenuate bone loss and reduce bone marrow fat by preventing bone marrow endothelial-to-adipocyte transition. |
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