| purpose:To explore the mechanism of α-klotho affecting cognitive function in CKD mice,and to further explore the possible pathway and mechanism of exogenous α-klotho protein improving cognitive function in CKD mice.Materials and Methods:CKD mouse was established by ligation of the main branch of the left renal artery and right nephrectomy in adult mice.Ninety-five mice were randomly divided into 5groups: normal group(n=17),normal +klotho group(n=20),sham operation group(n=17),CKD group(n=20),CKD+Klotho group(n= 21).Mice in CKD+klotho group and normal +klotho group were intraperitoneally injected with recombinant Klotho protein 0.01mg/kg once every two days for four weeks.Mice in the other groups were injected with PBS according to body weight.After 8 weeks,cerebrospinal fluid(CSF)was collected to detect the concentration of α-klotho protein with His tag by Elisa.The concentrations of SYP and PSD95 in hippocampus homogenate were detected by Elisa.Transmission electron microscopy was used to observe synaptic ultrastructure changes in hippocampal CA1 region.Immunoelectron microscopy was used to observe whether exogenous klotho could cross the blood-brain barrier and its distribution around the blood-brain barrier.Result:1.Elisa experiment:(1)Concentration of His-tagged klotho protein in cerebrospinal fluid: The normal group(15.91±4.249 pg/ml),normal +klotho group(13.85±4.226 pg/ml),sham operation group(13.59±5.368 pg/ml)and CKD group(13.56±5.615 pg/ml)showed no significant difference(P > 0.05).CKD+klotho group(122.8±55.43 pg/ml)was significantly higher than other groups(P < 0.05).(2)Hippocampal SYP concentration: Compared with the normal group(173.9±12.87ng/ml)and the sham operation group(180.2±12.49 ng/ml),the CKD group(146.2±13.95ng/ml)was significantly decreased(P < 0.05).Compared with CKD group(176.6±21.97ng/ml),normal +klotho group(191.7±9.294 ng/ml)was significantly increased(P <0.05).(3)PSD95 concentration in hippocampus: Compared with the normal group(95.21±15.32 ng/ml)and sham operation group(89.39±23.74 ng/ml),the level of PSD95 protein in hippocampus of CKD group(48.59±11.03ng/ml)was significantly decreased(P < 0.05).CKD+klotho group(67.60±7.677 ng/ml)was still lower than normal group(P < 0.05),but still higher than CKD group(P < 0.05).The expression ofα-klotho protein in normal +klotho group(132.9±26.72 ng/ml)was significantly higher than that in normal group(P < 0.05).(2)HE staining: there was no obvious degeneration of hippocampal neurons in normal group,normal +klotho group and sham operation group.The brain tissue structure of CKD group was abnormal,and a large number of hippocampal neurons degenerated.3.Hippocampal synaptic ultrastructure:(1)Synaptic density: There was no significant difference between the normal group(29.81±2.571/um2)and the shamoperated group(19.68±4.094/um2)(P > 0.05).Compared with the normal group,the normal +klotho group(39.17±1.703/um2)had a significant increase in synaptic density(P < 0.05).The synaptic density in CKD group(19.68±4.094/um2)was significantly lower than that in normal group and sham-operated group(P < 0.05).The synaptic density in CKD+klotho group(24.79±3.084/um2)was significantly higher than that in CKD group(P < 0.05).(2)Width of hippocampal CA1 synaptic cleft: There was no significant difference between the normal group(14.09±2.148nm)and the shamoperated group(14.16±2.01 nm)(P > 0.05).Compared with the normal group,the normal +klotho group(12.00±1.534 nm)had a significantly narrower synaptic cleft width(P < 0.05).The width of synaptic cleft in CKD mice(16.59±1.1844 nm)was significantly increased(P < 0.05),and the width of synaptic cleft in CKD+klotho group(15.18±1.1877 nm)was narrower than that in CKD group(P > 0.05),but there was still a difference between CKD+ Klotho group and normal group and sham group(P < 0.05).(3)hippocampal CA1 postsynaptic membrane thickness: There was no significant difference in the thickness of synaptic density between the normal group(15.42±3.661nm)and the sham-operated group(16.36±4.056 nm)(P > 0.05).The nm in normal+klotho group(20.91±3.566 nm)was significantly higher than that in normal group(P <0.05),and the nm in CKD group(12.85±3.100 nm)was significantly lower than that in normal group and sham group(P < 0.05).Compared with CKD group,CKD+klotho group(14.97±3.308 nm)increased significantly(P < 0.05).4.Hippocampal synaptic blood-brain barrier immunoelectron microscopy: In normal +klotho group,colloidal gold ions were scattered around the blood-brain barrier,and in CKD+klotho group,colloidal gold ions were distributed around the blood-brain barrier with opening of the tight junction of the blood-brain barrier and blurring of the basement membrane.4.Immune electron microscopy of blood-brain barrier at hippocampal synapse: In normal+klotho group,colloidal gold ions were scattered around the blood-brain barrier,and in CKD+klotho group,colloidal gold ions were distributed around the blood-brain barrier with opening of the tight junction of the blood-brain barrier and blurring of the basement membrane.Conclusion:1.CKD leads to degeneration of hippocampal neurons in mice,reduced synaptic density and changes in synaptic plasticity(including widening of synaptic gap and reduced thickness of postsynaptic dense matter),which may be an important biological basis for cognitive impairment in CKD mice.2.Exogenous alpha-Klotho protein may improve cognitive impairment in CKD mice by reducing hippocampal neuronal degeneration and enhancing hippocampal synaptic plasticity.3.Exogenous alpha-Klotho protein can cross the blood-brain barrier and directly enter cerebrospinal fluid to play its biological role in CKD.4.Interestingly,we found in our study that although exogenous α-klotho protein could not cross the blood-brain barrier in healthy mice,it could still effectively improve hippocampal synaptic plasticity,and its mechanism is unknown,which is worthy of further study. |
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