Research And Exploration Based On The Early Diagnosis Of Renal Fibrosis

Part 1 Population-based study of diagnostic indicators of renal fibrosisObjective:By analyzing the indexes that reflect the severity of renal fibrosis in patients with different stages of chronic kidney disease(CKD),we further screened the indexes that can better reflect the severity of renal fibrosis and have diagnostic value by comparing the differences between groups,ordered logistic regression analysis,and ROC curves.Objects and Methods:Patients with CKD who were hospitalized in the hospital from January 25,2022 to January 25,2023 were collected using the physician workstation and electronic medical record system of Baiqiuen Hospital in Shanxi Province.Stratified random sampling was used for sampling,and firstly,CKD patients were divided into three groups according to the clinical stage of CKD,one group for stage 1-2,one group for stage 3-4,and one group for stage 5;then 49 patients were selected from each group of CKD patients using simple random sampling method,for a total of 147 patients as study subjects.Fifteen indexes such as patients’ gender,age,BUN,Scr,uric acid,homocysteine,natriuretic peptide,urinary β2 microglobulin,ALB,triglycerides,TNF-α,IL-6,IFN-γ,RBP,and Hs-CRP were collected.ANOVA and chi-square test were used to analyze whether there were differences in the indicators of different stages of CKD,ordered logistic regression model was used to analyze the association between CKD and its related diagnostic indicators,and finally the diagnostic value of each group of indicators was predicted by ROC curve.Results:Among 147 patients with CKD,males were the most represented in the stage 1-2 group,stage 3-4 group and stage 5 group with 63.27%,53.06%and 61.22%,respectively,and patients in the stage 5 group were older compared to stage 1-2 and stage 3-4 with a maximum age of(60.00 ± 2.20)years.The differences in 12 indicators,including age,BUN,Scr,uric acid,homocysteine,natriuretic peptide,ALB,TNF-α,IL-6,IFN-γ,RBP,and Hs-CRP,were statistically significant between the groups(P<0.05).The results of ordered logistic regression analysis showed that high urea(OR=1.323,95%CI:1.230,1.424),high blood creatinine(OR=1.019,95%CI:1.013,1.024),high uric acid(OR=1.006,95%CI:1.628,4.795),high homocysteine(OR=1.000,95%CI:1.628,4.795),high natriuretic peptide(OR=1.002,95%CI:1.001,1.003),high urinary 32 microglobulin(OR=2.513,95%CI:1.856,3.403),high TNF-α(OR=7.102,95%CI:4.489,11.237),high IL-6(OR=1.237,95%CI:1.171,1.307),high IFN-γ(OR=9.477,95%CI:4.616,19.458),high RBP(OR=1.017,95%CI:1.008,1.026),high Hs-CRP(OR=1.019,95%CI:1.010,1.028)were independent risk factors for the severity of CKD.In the ROC curve analysis,no indicators in the stage 1-2 group had diagnostic value for CKD(P<0.05,AUC<0.5);again,no indicators in the stage 3-4 group had diagnostic value for CKD(P>0.05);in the stage 5 group,BUN,Scr,uric acid,natriuretic peptide,urinary β2 microglobulin,TNF-α,IL-6,IFN-γ,RBP,and Hs-CRP all had diagnostic value for CKD had diagnostic value(P<0.05,AUC>0.5).Conclusion:This study showed that elevated BUN,Scr,uric acid,homocysteine,natriuretic peptide,urinary β2-microglobulin,TNF-α,IL-6,IFN-γ,RBP and Hs-CRP were associated with CKD severity and were independent risk factors for CKD.In the stage 1-2 group,there were no indicators of diagnostic value in the stage 3-4 group,and most indicators(BUN,Scr,uric acid,natriuretic peptide,urinary β2 microglobulin,TNF-α,IL-6,IFN-y,RBP,and Hs-CRP)were of diagnostic value in the stage 5 group.Since the progression pathway of CKD is reached through renal fibrosis,the indexes reflecting different severity of CKD also correlate with the severity of renal fibrosis and can be used as independent risk factors for renal fibrosis.Although indicators of independent risk factors for renal fibrosis were screened,these indicators only have diagnostic significance for stage 5 patients,and the association between these indicators and renal fibrosis still needs to be further verified by a large number of samples and pathological tissues of different stages of CKD.In conclusion,the exploration of indicators that can reflect the severity of early renal fibrosis is extremely important for the early diagnosis of CKD.Part 2 Synthesis and properties of MNP-PEG-MnObjective:In this study,we designed and synthesized a water-soluble MNP-PEG-Mn nanoparticle with a particle size of about 2.7 nm,which can be used for multi-modal in vivo diagnostic imaging of early renal fibrosis by magnetic resonance imaging(MRI)and photoacoustic imaging(PAI).Methods:1.Synthesis and characterization of MNP-PEG-Mn① MNP nanoprobes were prepared,surface modified with 5000 molecular weight polyethylene glycol,and subsequently reacted with aqueous MnCl2 solution with stirring to synthesize MNP-PEG-Mn;② probe morphology and particle size were observed using transmission electron microscopy(TEM);fluid particle size as well as electronegativity were determined using DLS;absorption in UV-Vis-NIR was measured using UV spectrophotometer;and FT-IR infrared spectroscopy to verify the normal connection between MNP and PEG;the stability of the Mn2+ connection in MNP-PEG-Mn was checked by dialysis experiments.2.In vitro performance evaluation① Solution level:observe the anti-free radical ability of MNP-PEG-Mn;prepare different concentrations of MNP-PEG-Mn for MRI and PAI of the solution;② cellular level:observe the survival rate of cells after incubation with NRK-52E at different concentrations of MNP-PEG-Mn for 24 h using CCK-8;incubate MNP-PEG-Mn with NRK-52E for 5 h at one-hour intervals to observe the labeling rate by flow detection;observe the uptake and distribution of MNP-PEG-Mn in cells indirectly and directly using confocal and cellular electron microscopy,respectively.3.In vivo performance evaluationIn vivo imaging of photoacoustics and NMR was performed in normal group,7 days group of renal fibrosis model,and 28 days group of renal fibrosis model,respectively.Signal images were collected before and 2 h,4 h,6 h,12 h and 24 h after administration of MNP-PEG-Mn in each group,and quantitative analysis was done.4.Section experiments and blood biochemical experimentsH&E and Masson staining,blood creatinine and urea nitrogen were collected from normal group,7 days renal fibrosis group and 28 days renal fibrosis group,and the changes in each group were analyzed by immunohistochemistry for α-SMA,Collagen I and Fibronectin proteins highly expressed in fibrous tissues,and compared with clinical biochemical tests and the pathological methods were compared with clinical biochemical tests and pathology.5.Safety experiments of MNP-PEG-MnThe major organs(heart,liver,spleen,lung and kidney)of normal mice injected with and without MNP-PEG-Mn for 24 h,as well as the blood creatinine,urea nitrogen,alanine transaminase and glutathione transaminase from both groups were collected and analyzed by H&E for histological staining,and biochemical analysis to further confirm the biosafety of MNP-PEG-Mn.Results:1.The MNP-PEG-Mn was successfully synthesized,and the transmission electron microscope observed that the nanoprobe was round and uniformly distributed;the fluid particle size of MNP-PEG-Mn was 2.7 ± 2.2 nm with negative charge measured by DLS;the UV spectrum of MNP-PEG-Mn showed absorption in the range of 200-1000 nm,and the light absorption of the probe showed a slope decrease.The position of the characteristic peak of melanin was at 3500 cm-1 and that of PEG was at 500 cm-1.The PEG-modified MNP had two peaks,which could prove the successful modification of PEG onto the MNP surface.3%Mn2+ was released from MNP-PEG-Mn in the first 2 h.The release reached a plateau after 2 h,and did not continue to release Mn2+ in the following 48 h.2.①Solution level:both MR and PA signal intensities of MNP-PEG-Mn solution increased linearly with the increase of probe concentration,and the strongest signal was observed at 1500 μg/mL;② cellular level:CCK-8 results showed that the survival rate of NRK-52E was as high as 95%after 24 h co-incubation of MNP-PEG-Mn with NRK-52E,even if the probe concentration was as high as 1500 μg/mL.The cell identification by flow cytometry showed that the uptake of MNP-PEG-Mn by NRK-52E had reached 99%at 2 h incubation;the green fluorescence of FITC-MNP-PEG-Mn was observed in the cytoplasm around the blue nucleus stained by DAPI by laser confocal;the cell TEM results showed the presence of black nanoparticles in the cytoplasm.3.After tail vein injection of MNP-PEG-Mn,all groups reached signal maxima at 6 h.The 7 days group of renal fibrosis mice showed the strongest photoacoustic and nuclear magnetic signals,after which the normal group metabolized MNP-PEG-Mn completely at 24 h.Both the 7 days group of renal fibrosis mice and the 28 days group of renal fibrosis mice had retention in the kidney,but the metabolic curve of the 28 days group were flatter.4.It was clearly seen in the 7 days and 28 days groups that the fibrotic area was greater in the 28 days group compared to the normal group than in the 7 days group.Also blood biochemical results showed a significant increase in urea nitrogen by about 2.74-fold and blood creatinine by about 2.32-fold in the 28 days group compared to the normal group.Immunohistochemical analysis showed that α-SMA,collagen I and fibronectin were significantly increased compared to the normal group.5.No significant pathological changes,tissue destruction or inflammatory infiltration were observed in the tissue structures of all major organs in both groups of mice.In addition,in the collected blood creatinine,urea nitrogen,alanine transaminase and glutathione transaminase,serum biochemical analysis showed no significant changes in each index.Conclusion:This topic successfully developed a novel ultrasmall MNP-PEG-Mn nanoplatform for 7 days renal fibrosis diagnosis that can be used as a safe,non-toxic,biocompatible diagnostic contrast agent for PA/MRI dual-mode imaging.The obtained ultra-small size(≈2.7 nm)of MNP-PEG-Mn can be passively targeted to accumulate into the kidney through its positive charge effect and ultra-small particle size.As we expected,MNP-PEG-Mn showed much higher PA and MRI signals in the 7 days renal fibrosis group than in the normal and 28-days intravenous groups,and due to its good anti-free radical ability,MNP did not further contribute to the progression of renal disease.Therefore,MNP-PEG-Mn with ultra-small particle size provides a novel nano-contrast agent with good clinical application for overcoming the accurate diagnosis of early chronic kidney disease.