Effect And Mechanism Of A Novel Phosphate Binder:Amino-Modified Cellulose On Osteoporosis In Rats With Chronic Kidney Disease

Objective All chronic kidney disease?CKD?end-stage patients can develop mineral and bone Bone disorder,and osteoporosis is the most common manifestation of abnormal bone metabolism in CKD,mainly characterized by bone mass reduction and bone microstructural destruction.Studies have shown that hyperphosphatemia can occur in CKD patients at an early stage,which is the initiation factor and important link of CKD osteoporosis,which can directly stimulate the secretion of parathyroid hormone?PTH?and enhance the osteoclastic effect.In addition,hyperphosphatemia can lead to hyperlipidemia in cell metabolic disorders,and blood lipid levels are inversely related to bone strength and are an independent risk factor for osteoporosis.Recent studies have shown that the osteoprotectin/the receptor activator of nuclear factor?B ligand/receptor activator of NF-?B?OPG/RANKL/RANK?system pathway plays a key role in regulating bone metabolism,which is the most important pathway to regulate osteoclast activation.Amino-modified cellulose?AMC?is a novel phosphorus binder independently developed by using cellulose as the substrate,our results of previous study have shown that AMC can effectively reduce blood phosphorus levels in CKD rats.However,whether it can improve osteoporosis in CKD rats is not clear.In this study,the model of osteoporosis in CKD rats was established by 5/6 nephrectomy combined with high-phosphorus feed,to investigate the effect and mechanism of AMC on osteoporosis in CKD rats,and provide a new experimental methods and theoretical basis for the clinical application of AMC in the treatment and improvement of osteoporosis in CKD patients.Methods Forty-two clean-grade healthy male SD rats,aged 8 weeks,weighing 160-180 g.A random digital table was used to divide into seven groups?n=6?:sham group?group S?,model group?group M?,calcium carbonate group?CaCO₃,group C?,sevelamer group?reagel?,group R?,low-dose AMC group?group AMC-LD?,medium-dose AMC group?group AMC-MD?and high-dose AMC group?group AMC-HD?.The model of osteoporosis in CKD rats was established by 5/6 nephrectomy combined with high-phosphorus feed.At 6weeks after establishing the model,each drug group was given intragastrically at 9:00 a.m.every day,once a day.At 6 weeks after establishing the model and 4 weeks after administration,the blood sample were collected from the caudal cusp vein under sevoflurane inhalation anesthesia.Serum creatinine?Cr?,blood urea nitrogen?BUN?,total cholesterol?TC?and triglyceride?TG?level was measured by biochemical;Serum intact parathyroid hormone?iPTH?level was detected by ELISA;The BMD value of the whole body was measured by dual energy x-ray.At 4 weeks after administration,serum bone alkaline phosphatase?BALP?level was detected by ELISA and serum anti-tarte acid phosphatase 5b?TRAP5b?level was detected by microplate method;Rats were sacrificed by abdominal aortic puncture and bleeding under sevoflurane inhalation anesthesia,kidney tissue and tibia tissue of rats were collected to prepare paraffin sections for H&E staining,the pathological changes were observed and histomorphometric analysis was performed;Tibial tissue sections were stained with TRAP to determine the number of osteoclasts;Immunohistochemistry?IHC?was used to detect the expression levels of OPG and RANKL in tibial tissue;Femoral tissue of rats were collected and the expression levels of OPG and RANKL mRNA were detected by RT-PCR.Results At 6 weeks after establishing the model,compared with the group S,the levels of serum Cr,BUN,TC,TG and iPTH in the other groups were significantly increased?P<0.01?,and the whole body BMD was significantly decreased?P<0.01?.At 4 weeks after administration,compared with the group M,the levels of serum BUN,TC,TG,BALP,TRAP5b and iPTH in the groups R,C,AMC-LD,AMC-MD and AMC-HD were significantly reduced?P<0.01?,in addition to these serum indicators,the whole body BMD increased significantly?P<0.01?,the percentage of glomerular area?%?and the diameter of glomeruli?mm?decreased significantly?P<0.01?,the percentage of area of TRAP-positive cells?%?,bone trabecular separation?Tb.SP?and RANKL expression level in tibial tissue were decreased significantly?P<0.01?,OPG and RANKL mRNA expression levels in femur were significantly reduced?P<0.01?.In the group C and group AMC-HD the percentage of glomerular cavity space area?%?were decreased?P<0.05?.In the groups C,R,AMC-MD and AMC-HD,the trabecular thickness?Tb.Th?were increased?P<0.05?,the percentage of trabecular bone area?Tb.Ar%?and the percentage of cortical bone area?Ct.Ar%?increased significantly,and the expression level of OPG increased significantly?P<0.01?.At 4 weeks after administration,compared with group C,the levels of serum TC in group R and group AMC-HD were significantly reduced?P<0.01?,and the levels of TG in group R,group AMC-MD and group AMC-HD were reduced?P<0.05?;The area percentage?%?of TRAP-positive cells in the AMC-LD group increased significantly,and the expression level of OPG decreased significantly?P<0.01?;The expression levels of RANKL in group R,group AMC-LD and group AMC-MD were significantly increased?P<0.01?;group AMC-HD had a significant increase in systemic BMD?P<0.01?.At 4 weeks after administration,compared with the group R,the levels of serum BUN,TRAP5b,and iPTH in the group AMC-HD were reduced?P<0.05?,the expression level of RANKL was significantly reduced?P<0.01?,and systemic BMD was significantly increased?P<0.01?.Conclusion AMC can effectively improve osteoporosis in CKD rats with a dose-dependent manner.The mechanism may be related to the reduction of serum TC?TG and iTPH levels in CKD rats and down-regulation of expression of the OPG/RANKL/RANK system pathway,thereby inhibiting osteoclastogenesis.